Mutant G-protein coupled receptors and methods for selecting them

ABSTRACT

The invention relates to mutant G-protein coupled receptors with increased conformational stability, and methods of use thereof. In some aspects, polynucleotides encoding the mutant G-protein coupled receptors are provided. In some aspects, host cells comprising the polynucleotides are provided. In some aspects, the invention relates to crystallized forms of the mutant G-protein coupled receptors, and methods of preparing the same.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/215,533 filed Dec. 10, 2018, entitled “Mutant G-Protein CoupledReceptors and Methods For Selecting Them,” which is a continuation ofU.S. patent application Ser. No. 15/716,302 filed Sep. 26, 2017,entitled “Mutant G-Protein Coupled Receptors and Methods For SelectingThem,” which is a continuation of U.S. patent application Ser. No.14/836,820 filed Aug. 26, 2015, entitled “Mutant G-Protein CoupledReceptors and Methods For Selecting Them,” which is a continuation ofU.S. patent application Ser. No. 13/493,898, filed Jun. 11, 2012,entitled “Mutant G-Protein Coupled Receptors and Methods For SelectingThem,” which is a continuation of U.S. patent application Ser. No.14/450,358, filed Mar. 19, 2010, entitled “Mutant G-Protein CoupledReceptors and Methods For Selecting Them,” now U.S. Pat. No. 8,785,135,which is a national stage filing under 35 U.S.C. § 371 of internationalapplication PCT/GB2008/000986, filed Mar. 20, 2008, which was publishedunder PCT Article 21(2) in English and claims priority to UK PatentApplication No. 0724052.6, filed Dec. 8, 2007, and UK Patent ApplicationNo. 0705450.5, filed Mar. 22, 2007, the entire disclosures of each ofwhich are herein incorporated by reference in their entireties.

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The present invention relates to mutant G protein coupled receptors(GPCRs) and methods for selecting those with increased stability. Inparticular, it relates to the selection and preparation of mutant GPCRswhich have increased stability under a particular condition compared totheir respective parent proteins. Such proteins are more likely to becrystallisable, and hence amenable to structure determination, than theparent proteins. They are also useful for drug discovery and developmentstudies.

Over the past 20 years the rate of determination of membrane proteinstructures has gradually increased, but most success has been incrystallising membrane proteins from bacteria rather than fromeukaryotes [1]. Bacterial membrane proteins have been easier tooverexpress using standard techniques in Escherichia coli thaneukaryotic membrane proteins [2,3] and the bacterial proteins aresometimes far more stable in detergent, detergent-stability being anessential prerequisite to purification and crystallisation. Genomesequencing projects have also allowed the cloning and expression of manyhomologues of a specific transporter or ion channel, which also greatlyimproves the chances of success during crystallisation. However, out ofthe 120 different membrane protein structures that have been solved todate, there are only seven structures of mammalian integral membraneproteins (blanco.biomol.uci.edu/); five of these membrane proteins werepurified from natural sources and are stable in detergent solutions.Apart from the difficulties in overexpressing eukaryotic membraneproteins, they often have poor stability in detergent solutions, whichseverely restricts the range of crystallisation conditions that can beexplored without their immediate denaturation or precipitation. Ideally,membrane proteins should be stable for many days in any given detergentsolution, but the detergents that are best suited to growingdiffraction-quality crystals tend to be the most destabilisingdetergents ie those with short aliphatic chains and small or chargedhead groups. It is also the structures of human membrane proteins thatwe would like to solve, because these are required to help thedevelopment of therapeutic agents by the pharmaceutical industry; oftenthere are substantial differences in the pharmacology of receptors,channels and transporters from different mammals, whilst yeast andbacterial genomes may not include any homologous proteins. There is thusan overwhelming need to develop a generic strategy that will allow theproduction of detergent-stable eukaryotic integral membrane proteins forcrystallisation and structure determination and potentially for otherpurposes such as drug screening, bioassay and biosensor applications.

Membrane proteins have evolved to be sufficiently stable in the membraneto ensure cell viability, but they have not evolved to be stable indetergent solution, suggesting that membrane proteins could beartificially evolved and detergent-stable mutants isolated [4]. This wassubsequently demonstrated for two bacterial proteins, diacylglycerolkinase (DGK) [5,6] and bacteriorhodopsin [7]. Random mutagenesis of DGKidentified specific point mutations that increased thermostability and,when combined, the effect was additive so that the optimally stablemutant had a half-life of 35 minutes at 80° C. compared with a half-lifeof 6 minutes at 55° C. for the native protein [6]. It was shown that thetrimer of the detergent-resistant DGK mutant had become stable in SDSand it is thus likely that stabilisation of the oligomeric state playeda significant role in thermostabilisation. Although the aim of themutagenesis was to produce a membrane protein suitable forcrystallisation, the structure of DGK has yet to be determined and therehave been no reports of successful crystallization. A further study onbacteriorhodopsin by cysteine-scanning mutagenesis along helix Bdemonstrated that it was not possible to predict which amino acidresidues would lead to thermostability upon mutation nor, when studiedin the context of the structure, was it clear why thermostabilisationhad occurred [7].

GPCRs constitute a very large family of proteins that control manyphysiological processes and are the targets of many effective drugs.Thus, they are of considerable pharmacological importance. A list ofGPCRs is given in Foord et al (2005) Pharmacol Rev. 57, 279-288, whichis incorporated herein by reference. GPCRs are generally unstable whenisolated, and despite considerable efforts, it has not been possible tocrystallise any except bovine rhodopsin, which naturally isexceptionally stable.

GPCRs are druggable targets, and reference is made particularly toOverington et al (2006) Nature Rev. Drug Discovery 5, 993-996 whichindicates that over a quarter of present drugs have a GPCR as a target.

GPCRs are thought to exist in multiple distinct conformations which areassociated with different pharmacological classes of ligand such asagonists and antagonists, and to cycle between these conformations inorder to function (Kenakin T. (1997) Ann NY Acad Sci 812, 116-125).

It will be appreciated that the methods of the invention do not includea method as described in D'Antona et al., including binding of[³H]CP55940 to a constitutively inactive mutant human cannabinoidreceptor 1 (T210A) in which the Thr residue at position 210 is replacedwith an Ala residue.

The listing or discussion of an apparently prior-published document inthis specification should not necessarily be taken as an acknowledgementthat the document is part of the state of the art or is common generalknowledge.

We have realised that there are two serious problems associated withtrying to crystallise GPCRs, namely their lack of stability in detergentand the fact that they exist in multiple conformations. In order tofunction GPCRs have evolved to cycle through at least two distinctconformations, the agonist-bound form and the antagonist-bound form, andchanges between these two conformations can occur spontaneously in theabsence of ligand. It is thus likely that any purified receptorspopulate a mixture of conformations. Just adding ligands to GPCRs duringcrystallisation trials has not resulted in their structuredetermination. To improve the likelihood of crystallisation, wetherefore selected mutations that improved the stability of the GPCRand, in addition, preferentially locked the receptor in a specificbiologically relevant conformation.

We decided to see whether stabilisation of a GPCR in a particular,biologically relevant conformation was possible and whether the effectwas sufficiently great that it would significantly improve the chancesof obtaining diffraction-quality crystals. In Example 1, theβ1-adrenergic receptor (βAR) from turkey erythrocytes [8] was chosen asa test subject for this study for a number of reasons. The βAR is a Gprotein-coupled receptor (GPCR) that has well-developed pharmacologywith many ligands commercially available and in a radiolabelled form. Inaddition, overexpression of βAR has been particularly successful usingthe baculovirus expression system and it can be purified in milligramquantities in a functional form [9]. In Example 2, a human adenosinereceptor was used, and in Example 3, a rat neurotensin receptor wasused.

Method for Selecting Mutant GPCRs with Increased Stability

A first aspect of the invention provides a method for selecting a mutantG-protein coupled receptor (GPCR) with increased stability, the methodcomprising

-   -   (a) providing one or more mutants of a parent GPCR,    -   (b) selecting a ligand, the ligand being one which binds to the        parent GPCR when the GPCR is residing in a particular        conformation,    -   (c) determining whether the or each mutant GPCR has increased        stability with respect to binding the selected ligand compared        to the stability of the parent GPCR with respect to binding that        ligand, and    -   (d) selecting those mutants that have an increased stability        compared to the parent GPCR with respect to binding of the        selected ligand.

The inventors have appreciated that, in order to improve the likelihoodof crystallisation of a GPCR in a biologically relevant form (which istherefore pharmacologically useful), it is desirable not only toincrease the stability of the protein, but also for the protein to havethis increased stability when in a particular conformation. Theconformation is determined by a selected ligand, and is a biologicallyrelevant conformation in particular a pharmacologically relevantconformation. Thus, the method of the invention may be considered to bea method for selecting mutants of a GPCR which have increased stabilityof a particular conformation, for example they may have increasedconformational thermostability. The method may be used to create stable,conformationally locked GPCRs by mutagenesis. The selected mutant GPCRsare effectively purer forms of the parent molecules in that a muchhigher proportion of them occupies a particular conformational state.The deliberate selection of a chosen receptor conformation resolved fromother conformations by use of a ligand (or ligands) that bindpreferentially to this conformation is therefore an important feature ofthe invention. The method may also be considered to be a method forselecting mutant GPCRs which are more tractable to crystallisation.

Thus the invention includes a method for selecting a mutant G-proteincoupled receptor (GPCR) with increased stability, the method comprising

-   -   (a) providing one or more mutants of a parent GPCR,    -   (b) selecting a ligand, the ligand being one which binds to the        parent GPCR when the GPCR is residing in a particular        conformation,    -   (c) determining whether the or each mutant GPCR when residing in        the particular conformation has increased stability with respect        to binding the selected ligand compared to the stability of the        parent GPCR when residing in the same particular conformation        with respect to binding that ligand, and    -   (d) selecting those mutants that have an increased stability        compared to the parent GPCR with respect to binding of the        selected ligand.

In a review of the druggable genome by Hopkins & Groom (2002) NatureRev. Drug Discovery 1, 727-730, Table 1 contains a list of proteinfamilies many of which are GPCRs. Overington et al (2006) Nature Rev.Drug Discovery 5, 993-996 provides more details of drug targets, andFIG. 1 indicates that more than a quarter of current drugs target GPCRs.There are 52 GPCR targets for orally available drugs out of a total of186 total targets in this category.

Suitable GPCRs for use in the practice of the invention include, but arenot limited to β-adrenergic receptor, adenosine receptor, in particularadenosine A_(2a) receptor, and neurotensin receptor (NTR). Othersuitable GPCRs are well known in the art and include those listed inHopkins & Groom supra. In addition, the International Union ofPharmacology produce a list of GPCRs (Foord et al (2005) Pharmacol. Rev.57, 279-288, incorporated herein by reference and this list isperiodically updated at www.iuphar-db.org/GPCR/ReceptorFamiliesForward.It will be noted that GPCRs are divided into different classes,principally based on their amino acid sequence similarities. They arealso divided into families by reference to the natural ligands to whichthey bind. All GPCRs are included in the scope of the invention.

The amino acid sequences (and the nucleotide sequences of the cDNAswhich encode them) of many GPCRs are readily available, for example byreference to GenBank. In particular, Foord et al supra gives the humangene symbols and human, mouse and rat gene IDs from Entrez Gene(www.ncbi.nlm.nih.gov/entrez). It should be noted, also, that becausethe sequence of the human genome is substantially complete, the aminoacid sequences of human GPCRs can be deduced therefrom.

Although the GPCR may be derived from any source, it is particularlypreferred if it is from a eukaryotic source. It is particularlypreferred if it is derived from a vertebrate source such as a mammal ora bird. It is particularly preferred if the GPCR is derived from rat,mouse, rabbit or dog or non-human primate or man, or from chicken orturkey. For the avoidance of doubt, we include within the meaning of“derived from” that a cDNA or gene was originally obtained using geneticmaterial from the source, but that the protein may be expressed in anyhost cell subsequently. Thus, it will be plain that a eukaryotic GPCR(such as an avian or mammalian GPCR) may be expressed in a prokaryotichost cell, such as E. coli, but be considered to be avian- ormammalian-derived, as the case may be.

In some instances, the GPCR may be composed of more than one differentsubunit. For example, the calcitonin gene-related peptide receptorrequires the binding of a single transmembrane helix protein (RAMP1) toacquire its physiological ligand binding characteristics. Effector,accessory, auxiliary or GPCR-interacting proteins which combine with theGPCR to form or modulate a functional complex are well known in the artand include, for example, receptor kinases, G-proteins and arrestins(Bockaert et al (2004) Curr Opinion Drug Discov and Dev 7, 649-657).

The mutants of the parent GPCR may be produced in any suitable way andprovided in any suitable form. Thus, for example, a series of specificmutants of the parent protein may be made in which each amino acidresidue in all or a part of the parent protein is independently changedto another amino acid residue. For example, it may be convenient to makemutations in those parts of the protein which are predicted to bemembrane spanning. The three-dimensional structure of rhodopsin is known(Li et al (2004) J Mol Biol 343, 1409-1438; Palczewski et al (2000)Science 289, 739-745), and it is possible to model certain GPCRs usingthis structure. Thus, conveniently, parts of the GPCR to mutate may bebased on modelling. Similarly, computer programs are available whichmodel transmembrane regions of GPCRs based on hydrophobicity (Kyle &Dolittle (1982) J. Mol. Biol. 157, 105-132), and use can be made of suchmodels when selecting parts of the protein to mutate. Conventionalsite-directed mutagenesis may be employed, or polymerase chainreaction-based procedures well known in the art may be used. It ispossible, but less desirable, to use ribosome display methods in theselection of the mutant protein.

Typically, each selected amino acid is replaced by Ala (ie Ala-scanningmutagenesis), although it may be replaced by any other amino acid. Ifthe selected amino acid is Ala, it may conveniently be replaced by Leu.Alternatively, the amino acid may be replaced by Gly (ie Gly-scanningmutagenesis), which may allow a closer packing of neighbouring helicesthat may lock the protein in a particular conformation. If the selectedamino acid is Gly, it may conveniently be replaced by Ala.

Although the amino acid used to replace the given amino acid at aparticular position is typically a naturally occurring amino acid,typically an “encodeable” amino acid, it may be a non-natural amino acid(in which case the protein is typically made by chemical synthesis or byuse of non-natural amino-acyl tRNAs). An “encodeable” amino acid is onewhich is incorporated into a polypeptide by translation of mRNA. It isalso possible to create non-natural amino acids or introduce non-peptidelinkages at a given position by covalent chemical modification, forexample by post-translational treatment of the protein or semisynthesis.These post-translational modifications may be natural, such asphosphorylation, glycosylation or palmitoylation, or synthetic orbiosynthetic.

Alternatively, the mutants may be produced by a random mutagenesisprocedure, which may be of the whole protein or of a selected portionthereof. Random mutagenesis procedures are well known in the art.

Conveniently, the mutant GPCR has one replaced amino acid compared tothe parent protein (ie it is mutated at one amino acid position). Inthis way, the contribution to stability of a single amino acidreplacement may be assessed. However, the mutant GPCR assayed forstability may have more than one replaced amino acid compared to theparent protein, such as 2 or 3 or 4 or 5 or 6 replacements.

As is discussed in more detail below, combinations of mutations may bemade based on the results of the selection method. It has been foundthat in some specific cases combining mutations in a single mutantprotein leads to a further increase in stability. Thus, it will beappreciated that the method of the invention can be used in an iterativeway by, for example, carrying it out to identify single mutations whichincrease stability, combining those mutations in a single mutant GPCRswhich is the GPCR then provided in part (a) of the method. Thus,multiply-mutated mutant proteins can be selected using the method.

The parent GPCR need not be the naturally occurring protein.Conveniently, it may be an engineered version which is capable ofexpression in a suitable host organism, such as Escherichia coli. Forexample, as described in Example 1, a convenient engineered version ofthe turkey β-adrenergic receptor is one which is truncated and lacksresidues 1-33 of the amino acid sequence (ie βAR₃₄₋₄₂₄). The parent GPCRmay be a truncated form of the naturally occurring protein (truncated ateither or both ends), or it may be a fusion, either to the naturallyoccurring protein or to a fragment thereof. Alternatively oradditionally, the parent GPCR, compared to a naturally-occurring GPCR,may be modified in order to improve, for example, solubility,proteolytic stability (eg by truncation, deletion of loops, mutation ofglycosylation sites or mutation of reactive amino acid side chains suchas cysteine). In any event, the parent GPCR is a protein that is able tobind to the selected ligand which ligand is one which is known to bindthe naturally occurring GPCR. Conveniently, the parent GPCR is onewhich, on addition of an appropriate ligand, can affect any one or moreof the downstream activities which are commonly known to be affected byG-protein activation.

However, it will be appreciated that the stability of the mutant is tobe compared to a parent in order to be able to assess an increase instability.

A ligand is selected, the ligand being one which binds to the parentGPCR when residing in a particular conformation. Typically, the ligandwill bind to one conformation of the parent GPCR (and may cause the GPCRto adopt this conformation), but does not bind as strongly to anotherconformation that the GPCR may be able to adopt. Thus, the presence ofthe ligand may be considered to encourage the GPCR to adopt theparticular conformation. Thus, the method may be considered to be a wayof selecting mutant GPCRs which are trapped in a conformation ofbiological relevance (eg ligand bound state), and which are more stablewith respect to that conformation.

Preferably the particular conformation in which the GPCR resides in step(c) corresponds to the class of ligand selected in step (b).

Preferably the selected ligand is from the agonist class of ligands andthe particular conformation is an agonist conformation, or the selectedligand is from the antagonist class of ligands and the particularconformation is an antagonist conformation.

Preferably the selected ligand is from the agonist class of ligands andthe particular conformation in which the GPCR resides in step (c) is theagonist conformation. Preferably, the selected ligand binding affinityfor the mutant receptor should be equal to or greater than that for thewild type receptor; mutants that exhibit significantly reduced bindingto the selected ligand are typically rejected.

By “ligand” we include any molecule which binds to the GPCR and whichcauses the GPCR to reside in a particular conformation. The ligandpreferably is one which causes more than half of the GPCR moleculesoverall to be in a particular conformation.

Many suitable ligands are known.

Typically, the ligand is a full agonist and is able to bind to the GPCRand is capable of eliciting a full (100%) biological response, measuredfor example by G-protein coupling, downstream signalling events or aphysiological output such as vasodilation. Thus, typically, thebiological response is GDP/GTP exchange in a G-protein, followed bystimulation of the linked effector pathway. The measurement, typically,is GDP/GTP exchange or a change in the level of the end product of thepathway (eg cAMP, cGMP or inositol phosphates). The ligand may also be apartial agonist and is able to bind to the GPCR and is capable ofeliciting a partial (<100%) biological response.

The ligand may also be an inverse agonist, which is a molecule whichbinds to a receptor and reduces its basal (ie unstimulated by agonist)activity sometimes even to zero.

The ligand may also be an antagonist, which is a molecule which binds toa receptor and blocks binding of an agonist, so preventing a biologicalresponse. Inverse agonists and partial agonists may under certain assayconditions be antagonists.

The above ligands may be orthosteric, by which we include the meaningthat they combine with the same site as the endogenous agonist; or theymay be allosteric or allotopic, by which we include the meaning thatthey combine with a site distinct from the orthosteric site. The aboveligands may be syntopic, by which we include the meaning that theyinteract with other ligand(s) at the same or an overlapping site. Theymay be reversible or irreversible.

In relation to antagonists, they may be surmountable, by which weinclude the meaning that the maximum effect of agonist is not reduced byeither pre-treatment or simultaneous treatment with antagonist; or theymay be insurmountable, by which we include the meaning that the maximumeffect of agonist is reduced by either pre-treatment or simultaneoustreatment with antagonist; or they may be neutral, by which we includethe meaning the antagonist is one without inverse agonist or partialagonist activity. Antagonists typically are also inverse agonists.

Ligands for use in the invention may also be allosteric modulators suchas positive allosteric modulators, potentiators, negative allostericmodulators and inhibitors. They may have activity as agonists or inverseagonists in their own right or they may only have activity in thepresence of an agonist or inverse agonist in which case they are used incombination with such molecules in order to bind to the GPCR.

Neubig et al (2003) Pharmacol. Rev. 55, 597-606, incorporated herein byreference, describes various classes of ligands.

Preferably, the above-mentioned ligands are small organic or inorganicmoieties, but they may be peptides or polypeptides. Typically, when theligand is a small organic or organic moiety, it has a M_(r) of from 50to 2000, such as from 100 to 1000, for example from 100 to 500.

Typically, the ligand binds to the GPCR with a K_(d) of from mM to pM,such as in the range of from μM (micromolar) to nM. Generally, theligands with the lowest Kd are preferred.

Small organic molecule ligands are well known in the art, for examplesee the Examples below. Other small molecule ligands include 5HT whichis a full agonist at the 5HT1A receptor; eltoprazine which is a partialagonist at the 5HT1A receptor (see Newman-Tancredi et al (1997)Neurophamacology 36, 451-459); (+)-butaclamol and spiperone are dopamineD2 receptor inverse agonists (see Roberts & Strange (2005) Br. J.Pharmacol. 145, 34-42); and WIN55212-3 is a neutral antagonist of CB2(Savinainen et al (2005) Br. J. Pharmacol. 145, 636-645).

The ligand may be a peptidomimetic, a nucleic acid, a peptide nucleicacid (PNA) or an aptamer. It may be an ion such as Na⁺ or Zn²⁺, a lipidsuch as oleamide, or a carbohydrate such as heparin.

The ligand may be a polypeptide which binds to the GPCR. Suchpolypeptides (by which we include oligopeptides) are typically fromM_(r) 500 to M_(r) 50,000, but may be larger. The polypeptide may be anaturally occurring GPCR-interacting protein or other protein whichinteracts with the GPCR, or a derivative or fragment thereof, providedthat it binds selectively to the GPCR in a particular conformation.GPCR-interacting proteins include those associated with signalling andthose associated with trafficking, which often act via PDZ domains inthe C terminal portion of the GPCR.

Polypeptides which are known to bind certain GPCRs include any of a Gprotein, an arrestin, a RGS protein, G protein receptor kinase, a RAMP,a 14-3-3 protein, a NSF, a periplakin, a spinophilin, a GPCR kinase, areceptor tyrosine kinase, an ion channel or subunit thereof, an ankyrinand a Shanks or Homer protein. Other polypeptides include NMDA receptorsubunits NR1 or NR2a, calcyon, or a fibronectin domain framework. Thepolypeptide may be one which binds to an extracellular domain of a GPCR,such as fibulin-1. The polypeptide may be another GPCR, which binds tothe selected GPCR in a hetero-oligomer. A review of protein-proteininteractions at GPCRs is found in Milligan & White (2001) TrendsPharmacol. Sci. 22, 513-518, or in Bockaert et al (2004) Curr. OpinionDrug Discov. Dev. 7, 649-657 incorporated herein by reference.

The polypeptide ligand may conveniently be an antibody which binds tothe GPCR. By the term “antibody” we include naturally-occurringantibodies, monoclonal antibodies and fragments thereof. We also includeengineered antibodies and molecules which are antibody-like in theirbinding characteristics, including single chain Fv (scFv) molecules anddomain antibodies (dAbs). Mention is also made of camelid antibodies andengineered camelid antibodies. Such molecules which bind GPCRs are knownin the art and in any event can be made using well known technology.Suitable antibodies include ones presently used in radioimmunoassay(RIAs) for GPCRs since they tend to recognise conformational epitopes.

The polypeptide may also be a binding protein based on a modularframework, such as ankyrin repeat proteins, armadillo repeat proteins,leucine rich proteins, tetratriopeptide repeat proteins or DesignedAnkyrin Repeat Proteins (DARPins) or proteins based on lipocalin orfibronectin domains or Affilin scaffolds based on either human gammacrystalline or human ubiquitin.

In one embodiment of the invention, the ligand is covalently joined tothe GPCR, such as a G-protein or arrestin fusion protein. Some GPCRs(for example thrombin receptor) are cleaved N-terminally by a proteaseand the new N-terminus binds to the agonist site. Thus, such GPCRs arenatural GPCR-ligand fusions.

It will be appreciated that the use of antibodies, or other “universal”binding polypeptides (such as G-proteins which are known to couple withmany different GPCRs) may be particularly advantageous in the use of themethod on “orphan” GPCRs for which the natural ligand, and smallmolecule ligands, are not known.

Once the ligand has been selected, it is then determined whether the oreach mutant GPCR has increased stability with respect to binding theselected ligand compared to the parent GPCR with respect to binding thatligand. It will be appreciated that this step (c) is one in which it isdetermined whether the or each mutant GPCR has an increased stability(compared to its parent) for the particular conformation which isdetermined by the selected ligand. Thus, the mutant GPCR has increasedstability with respect to binding the selected ligand as measured byligand binding or whilst binding the selected ligand. As is discussedbelow, it is particularly preferred if the increased stability isassessed whilst binding the selected ligand.

The increased stability is conveniently measured by an extended lifetimeof the mutant under the imposed conditions which may lead to instability(such as heat, harsh detergent conditions, chaotropic agents and so on).Destabilisation under the imposed condition is typically determined bymeasuring denaturation or loss of structure. As is discussed below, thismay manifest itself by loss of ligand binding ability or loss ofsecondary or tertiary structure indicators.

As is described with respect to FIG. 12 below (which depicts aparticular, preferred embodiment), there are different assay formatswhich may be used to determine stability of the mutant GPCR.

In one embodiment the mutant GPCR may be brought into contact with aligand before being subjected to a procedure in which the stability ofthe mutant is determined (the mutant GPCR and ligand remaining incontact during the test period). Thus, for example, when the method isbeing used to select for mutant GPCRs which in one conformation bind toa ligand and which have improved thermostability, the receptor iscontacted with the ligand before being heated, and then the amount ofligand bound to the receptor following heating may be used to expressthermostability compared to the parent receptor. This provides a measureof the amount of the GPCR which retains ligand binding capacityfollowing exposure to the denaturing conditions (eg heat), which in turnis an indicator of stability.

In an alternative (but less preferred) embodiment, the mutant GPCR issubjected to a procedure in which the stability of the mutant isdetermined before being contacted with the ligand. Thus, for example,when the method is being used to select for mutant membrane receptorswhich in one conformation bind to a ligand and which have improvedthermostability, the receptor is heated first, before being contactedwith the ligand, and then the amount of ligand bound to the receptor maybe used to express thermostability. Again, this provides a measure ofthe amount of the GPCR which retains ligand binding capacity followingexposure to the denaturing conditions.

In both embodiments, it will be appreciated that the comparison ofstability of the mutant is made by reference to the parent moleculeunder the same conditions.

It will be appreciated that in both of these embodiments, the mutantsthat are selected are ones which have increased stability when residingin the particular conformation compared to the parent protein.

The preferred route may be dependent upon the specific GPCR, and will bedependent upon the number of conformations accessible to the protein inthe absence of ligand. In the embodiment described in FIG. 12 , it ispreferred if the ligand is present during the heating step because thisincreases the probability that the desired conformation is selected.

From the above, it will be appreciated that the invention includes amethod for selecting a mutant GPCR with increased thermostability, themethod comprising (a) providing one or more mutants of a parent GPCR,(b) selecting an antagonist or an agonist which binds the parent GPCR,(c) determining whether the or each mutant has increased thermostabilitywhen in the presence of the said antagonist or agonist by measuring theability of the mutant GPCR to bind the selected said antagonist oragonist at a particular temperature and after a particular time comparedto the parent GPCR and (d) selecting those mutant GPCRs that bind moreof the selected said antagonist or agonist at the particular temperatureand after the particular time than the parent GPCR under the sameconditions. In step (c), a fixed period of time at the particulartemperature is typically used in measuring the ability of the mutantGPCR to bind the selected said antagonist or agonist. In step (c),typically a temperature and a time is chosen at which binding of theselected said antagonist or agonist by the parent GPCR is reduced by 50%during the fixed period of time at that temperature (which is indicativethat 50% of the receptor is inactivated; “quasi” Tm).

Conveniently, when the ligand is used to assay the GPCR (ie used todetermine if it is in a non-denatured state), the ligand is detectablylabelled, eg radiolabelled or fluorescently labelled. In anotherembodiment, ligand binding can be assessed by measuring the amount ofunbound ligand using a secondary detection system, for example anantibody or other high affinity binding partner covalently linked to adetectable moiety, for example an enzyme which may be used in acolorimetric assay (such as alkaline phosphatase or horseradishperoxidase). FRET methodology may also be used. It will be appreciatedthat the ligand used to assay the mutant GPCR in determining itsstability need not be the same ligand as selected in step (b) of themethod.

Although it is convenient to measure the stability of the parent andmutant GPCR by using the ability to bind a ligand as an indicator of thepresence of a non-denatured protein, other methods are known in the art.For example, changes in fluorescence spectra can be a sensitiveindicator of unfolding, either by use of intrinsic tryptophanfluorescence or the use of extrinsic fluorescent probes such as1-anilino-8-napthaleneulfonate (ANS), for example as implemented in theThermofluor™ method (Mezzasalma et al, J Biomol Screening, 2007, April;12(3):418-428). Proteolytic stability, deuterium/hydrogen exchangemeasured by mass spectrometry, blue native gels, capillary zoneelectrophoresis, circular dichroism (CD) spectra and light scatteringmay also be used to measure unfolding by loss of signals associated withsecondary or tertiary structure. However, all these methods require theprotein to be purified in reasonable quantities before they can be used(eg high pmol/nmol quantities), whereas the method described in theExamples makes use of pmol amounts of essentially unpurified GPCR.

In a preferred embodiment, in step (b) two or more ligands of the sameclass are selected, the presence of each causing the GPCR to reside inthe same particular conformation. Thus, in this embodiment, one or moreligands (whether natural or non-natural) of the same class (eg fullagonist or partial agonist or antagonist or inverse agonist) may beused. Including multiple ligands of the same class in this process,whether in series or in parallel, minimises the theoretical risk ofinadvertently engineering and selecting multiply mutated receptorconformations substantially different to the parent, for example intheir binding site, but still able, due to compensatory changes, to bindligand. The following steps may be used to mitigate this risk:

1. Select a chemically distinct set (eg n=2-5) of ligands, in a commonpharmacological class as evidenced by for example a binding orfunctional or spectroscopic assay. These ligands should be thought tobind to a common spatial region of the receptor, as evidenced forexample by competitive binding studies using wild type and/or mutatedreceptors, and/or by molecular modelling, although they will notnecessarily express a common pharmacophore.

2. Make single or multiple receptor mutants intended to increasestability, and assay for tight binding using the full set of ligands.The assays can be parallelised, multiplexed or run in series.

3. Confirm authenticity of stabilised receptor mutant by measurement forexample of the binding isotherm for each ligand, and by measurement ofthe stability shift with ligand (the window should typically be narrowedcompared to wild type).

In order to guard against changes in apparent affinity caused byperturbations to the binding site upon mutation, preferably ligands ofthe same pharmacological class, but different chemical class, should beused to profile the receptor. These should typically show similar shiftsin affinity (mutant versus parent, e.g. wild type) in spite of havingdifferent molecular recognition properties. Binding experiments shouldpreferably be done using labelled ligand within the same pharmacologicalclass.

Nonetheless it should be recognised that conformational substrates mayexist that are specific to chemical classes of ligand within the samepharmacological class, and these may be specifically stabilised in theprocedure depending on the chemical class of the selected ligand.

Typically the selected ligand binds to the mutant GPCR with a similarpotency to its binding to the parent GPCR. Typically, the K_(d) valuesfor the particular ligand binding the mutant GPCR and the parent GPCRare within 5-10 fold of each other, such as within 2-3 fold. Typically,the binding of the ligand to the mutant GPCR compared to the parent GPCRwould be not more than 5 times weaker and not more than 10 timesstronger.

Typically, mutant receptors which have been stabilised in the selectedconformation should bind the selected ligand with approximately equalaffinity (that is to say typically within 2-3 fold) or greater affinitythan does the parent receptor. For agonist-conformation mutants, themutants typically bind the agonists with the same or higher affinitythan the parent GPCR and typically bind antagonists with the same orlower affinity than the parent GPCR. Similarly forantagonist-conformation mutants, the mutants typically bind theantagonists with the same or higher affinity than the parent GPCR andtypically bind agonists with the same or lower affinity than the parentGPCR.

Mutants that exhibit a significant reduction (typically greater than 2-3fold) in affinity for the selecting ligand are typically rejected.

Typically, the rank order of binding of a set of ligands of the sameclass are comparable, although there may be one or two reversals in theorder, or there may be an out-lier from the set.

In a further embodiment, two or more ligands that bind simultaneously tothe receptor in the same conformation may be used, for example anallosteric modulator and orthosteric agonist.

For the avoidance of doubt, and as is evident from the Examples, it isnot necessary to use multiple ligands for the method to be effective.

In a further embodiment, it may be advantageous to select those mutantGPCRs which, while still being able to bind the selected ligand, are notable to bind, or bind less strongly than the parent GPCR, a secondselected ligand which is in a different class to the first ligand. Thus,for example, the mutant GPCR may be one that is selected on the basisthat it has increased stability with respect to binding a selectedantagonist, but the mutant GPCR so selected is further tested todetermine whether it binds to a full agonist (or binds less strongly toa full agonist than its parent GPCR). Mutants are selected which do notbind (or have reduced binding of) the full agonist. In this way, furtherselection is made of a GPCR which is locked into one particularconformation.

It will be appreciated that the selected ligand (with respect to part(b) of the method) and the further (second) ligand as discussed above,may be any pair of ligand classes, for example: antagonist and fullagonist; full agonist and antagonist; antagonist and inverse agonist;inverse agonist and antagonist; inverse agonist and full agonist; fullagonist and inverse agonist; and so on.

It is preferred that the mutant receptor binds the further (second)ligand with an affinity which is less than 50% of the affinity theparent receptor has for the same further (second) ligand, morepreferably less than 10% and still more preferably less than 1% or 0.1%or 0.01% of affinity for the parent receptor. Thus, the K_(d) for theinteraction of the second ligand with mutant receptor is higher than forthe parent receptor. As is shown in Example 1, the mutant β-adrenergicreceptor βAR-m23 (which was selected by the method of the inventionusing an antagonist) binds an agonist 3 orders of magnitude more weaklythan its parent (ie K_(d) is 1000× higher). Similarly, in Example 2, themutant adenosine A2a receptor Rant21 binds agonist 2-4 orders ofmagnitude more weakly than its parent.

This type of counter selection is useful because it can be used todirect the mutagenesis procedure more specifically (and therefore morerapidly and more efficiently) along a pathway towards a pureconformation as defined by the ligand.

Preferably, the mutant GPCR is provided in a suitable solubilised formin which it maintains structural integrity and is in a functional form(eg is able to bind ligand). An appropriate solubilising system, such asa suitable detergent (or other amphipathic agent) and buffer system isused, which may be chosen by the person skilled in the art to beeffective for the particular protein. Typical detergents which may beused include, for example, dodecylmaltoside (DDM) or CHAPS oroctylglucoside (OG) or many others. It may be convenient to includeother compounds such as cholesterol hemisuccinate or cholesterol itselfor heptane-1,2,3-triol. The presence of glycerol or proline or betainemay be useful. It is important that the GPCR, once solubilised from themembrane in which it resides, must be sufficiently stable to be assayed.For some GPCRs, DDM will be sufficient, but glycerol or other polyolsmay be added to increase stability for assay purposes, if desired.Further stability for assay purposes may be achieved, for example, bysolubilising in a mixture of DDM, CHAPS and cholesterol hemisuccinate,optionally in the presence of glycerol. For particularly unstable GPCRs,it may be desirable to solubilise them using digitonin or amphipols orother polymers which can solubilise GPCRs directly from the membrane, inthe absence of traditional detergents and maintain stability typicallyby allowing a significant number of lipids to remain associated with theGPCR. Nanodiscs may also be used for solubilising extremely unstablemembrane proteins in a functional form.

Typically, the mutant GPCR is provided in a crude extract (eg of themembrane fraction from the host cell in which it has been expressed,such as E. coli). It may be provided in a form in which the mutantprotein typically comprises at least 75%, more typically at least 80% or85% or 90% or 95% or 98% or 99% of the protein present in the sample. Ofcourse, it is typically solubilised as discussed above, and so themutant GPCR is usually associated with detergent molecules and/or lipidmolecules.

A mutant GPCR may be selected which has increased stability to anydenaturant or denaturing condition such as to any one or more of heat, adetergent, a chaotropic agent or an extreme of pH.

In relation to an increased stability to heat (ie thermostability), thiscan readily be determined by measuring ligand binding or by usingspectroscopic methods such as fluorescence, CD or light scattering at aparticular temperature. Typically, when the GPCR binds to a ligand, theability of the GPCR to bind that ligand at a particular temperature maybe used to determine thermostability of the mutant. It may be convenientto determine a “quasi T_(m)” ie the temperature at which 50% of thereceptor is inactivated under stated conditions after incubation for agiven period of time (eg 30 minutes). Mutant GPCRs of higherthermostability have an increased quasi Tm compared to their parents.

In relation to an increased stability to a detergent or to a chaotrope,typically the GPCR is incubated for a defined time in the presence of atest detergent or a test chaotropic agent and the stability isdetermined using, for example, ligand binding or a spectroscopic methodas discussed above.

In relation to an extreme of pH, a typical test pH would be chosen (egin the range 4.5 to 5.5 (low pH) or in the range 8.5 to 9.5 (high pH).

Because relatively harsh detergents are used during crystallisationprocedures, it is preferred that the mutant GPCR is stable in thepresence of such detergents. The order of “harshness” of certaindetergents is DDM, C₁₁→C₁₀→C₉→C₈ maltoside or glucoside,lauryldimethylamine oxide (LDAO) and SDS. It is particularly preferredif the mutant GPCR is more stable to any of C₉ maltoside or glucoside,C₈ maltoside or glucoside, LDAO and SDS, and so it is preferred thatthese detergents are used for stability testing.

Because of its ease of determination, it is preferred thatthermostability is determined, and those mutants which have an increasedthermostability compared to the parent protein with respect to theselected condition are chosen. It will be appreciated that heat isacting as the denaturant, and this can readily be removed by cooling thesample, for example by placing on ice. It is believed thatthermostability may also be a guide to the stability to otherdenaturants or denaturing conditions. Thus, increased thermostability islikely to translate into stability in denaturing detergents, especiallythose that are more denaturing than DDM, eg those detergents with asmaller head group and a shorter alkyl chain and/or with a charged headgroup. We have found that a thermostable GPCR is also more stabletowards harsh detergents.

When an extreme of pH is used as the denaturing condition, it will beappreciated that this can be removed quickly by adding a neutralisingagent. Similarly, when a chaotrope is used as a denaturant, thedenaturing effect can be removed by diluting the sample below theconcentration in which the chaotrope exerts its chaotropic effect.

In a particular embodiment of the invention, the GPCR is β-adrenergicreceptor (for example from turkey) and the ligand is dihydroalprenolol(DHA), an antagonist.

In a further preferred embodiment of the invention, the GPCR is theadenosine A_(2a) receptor (A_(2a)R) (for example, from man) and theligand is ZM 241385 (4-[2-[[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-yl]amino]ethyl]phenol), an antagonist or NECA(5′-N-ethylcarboxamido adenosine), an agonist.

In a still further preferred embodiment, the GPCR is the neurotensinreceptor (NTR) (for example, from rat) and the ligand is neurotensin, anagonist.

A second aspect of the invention provides a method for preparing amutant GPCR, the method comprising

-   -   (a) carrying out the method of the first aspect of the        invention,    -   (b) identifying the position or positions of the mutated amino        acid residue or residues in the mutant GPCR or GPCRs which has        been selected for increased stability, and    -   (c) synthesising a mutant GPCR which contains a mutation at one        or more of the positions identified.

As can be seen in the Examples, surprisingly, changes to a single aminoacid within the GPCR may increase the stability of the protein comparedto the parent protein with respect to a particular condition in whichthe protein resides in a particular conformation. Thus, in oneembodiment of the method of the second aspect of the invention, a singleamino acid residue of the parent protein is changed in the mutantprotein. Typically, the amino acid residue is changed to the amino acidresidue found in the mutant tested in the method of the first aspect ofthe invention. However, it may be replaced by any other amino acidresidue, such as any naturally-occurring amino acid residue (inparticular, a “codeable” amino acid residue) or a non-natural aminoacid. Generally, for convenience, the amino acid residue is replacedwith one of the 19 other codeable amino acids. Preferably, it is thereplaced amino acid residue which is present in the mutant selected inthe first aspect of the invention.

Also as can be seen in the Examples, a further increase in stability maybe obtained by replacing more than one of the amino acids of the parentprotein. Typically, each of the amino acids replaced is one which hasbeen identified using the method of the first aspect of the invention.Typically, each amino acid identified is replaced by the amino acidpresent in the mutant protein although, as noted above, it may bereplaced with any other amino acid.

Typically, the mutant GPCR contains, compared to the parent protein,from 1 to 10 replaced amino acids, preferably from 1 to 8, typicallyfrom 2 to 6 such as 2, 3, 4, 5 or 6 replaced amino acids.

It will be appreciated that the multiple mutants may be subject to theselection method of the first aspect of the invention. In other words,multiple mutants may be provided in step (a) of the method of the firstaspect of the invention. It will be appreciated that by the first and/orsecond aspect of the invention multiply mutagenised GPCRs may be made,whose conformation has been selected to create a very stable multiplepoint mutant protein.

The mutant GPCRs may be prepared by any suitable method. Conveniently,the mutant protein is encoded by a suitable nucleic acid molecule andexpressed in a suitable host cell. Suitable nucleic acid moleculesencoding the mutant GPCR may be made using standard cloning techniques,site-directed mutagenesis and PCR as is well known in the art. Suitableexpression systems include constitutive or inducible expression systemsin bacteria or yeasts, virus expression systems such as baculovirus,semliki forest virus and lentiviruses, or transient transfection ininsect or mammalian cells. Suitable host cells include E. coli,Lactococcus lactis, Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pichia pastoris, Spodoptera frugiperda and Trichoplusiani cells.Suitable animal host cells include HEK 293, COS, S2, CHO, NSO, DT40 andso on. It is known that some GPCRs require specific lipids (egcholesterol) to function. In that case, it is desirable to select a hostcell which contains the lipid. Additionally or alternatively the lipidmay be added during isolation and purification of the mutant protein. Itwill be appreciated that these expression systems and host cells mayalso be used in the provision of the mutant GPCR in part (a) of themethod of the first aspect of the invention.

Molecular biological methods for cloning and engineering genes andcDNAs, for mutating DNA, and for expressing polypeptides frompolynucleotides in host cells are well known in the art, as exemplifiedin “Molecular cloning, a laboratory manual”, third edition, Sambrook, J.& Russell, D. W. (eds), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., incorporated herein by reference.

In a further embodiment of the first or second aspect of the inventionit is determined whether the selected or prepared mutant GPCR is able tocouple to a G protein. It is also preferred if it is determined whetherthe selected or prepared mutant GPCR is able to bind a plurality ofligands of the same class as the selecting ligand with a comparablespread and/or rank order of affinity as the parent GPCR.

A third aspect of the invention provides a mutant GPCR prepared by themethod of the second aspect of the invention.

The invention includes mutant GPCRs with increased stability compared totheir parent GPCRs, particularly those with increased thermostability.

Mutant β-adrenergic Receptor

β-adrenergic receptors are well known in the art. They share sequencehomology to each other and bind to adrenalin.

A fourth aspect of the invention provides a mutant β-adrenergic receptorwhich, when compared to the corresponding wild-type β-adrenergicreceptor, has a different amino acid at a position which corresponds toany one or more of the following positions according to the numbering ofthe turkey β-adrenergic receptor as set out in FIG. 9 : Ile 55, Gly 67,Arg 68, Val 89, Met 90, Gly 98, Ile 129, Ser 151, Val 160, Gln 194, Gly197, Leu 221, Tyr 227, Arg 229, Val 230, Ala 234, Ala 282, Asp 322, Phe327, Ala 334, Phe 338.

The mutant β-adrenergic receptor may be a mutant of any β-adrenergicreceptor provided that it is mutated at one or more of the amino acidpositions as stated by reference to the given turkey β-adrenergicreceptor amino acid sequence.

It is particularly preferred if the mutant GPCR is one which has atleast 20% amino acid sequence identity when compared to the given turkeyβ-adrenergic receptor sequence, as determined using MacVector andCLUSTALW (Thompson et al (1994) Nucl. Acids Res. 22, 4673-4680). Morepreferably, the mutant receptor has at least 30% or at least 40% or atleast 50% amino acid sequence identity. There is generally a higherdegree of amino acid sequence identity which is conserved around theorthosteric (“active”) site to which the natural ligand binds.

As is described in Example 1 and FIG. 1 below, individual replacement ofthe following amino acid residues in the parent turkey β-adrenergicsequence (as shown in FIG. 9 ) lead to an increase in thermostability:Ile 55, Gly 67, Arg 68, Val 89, Met 90, Gly 98, Ile 129, Ser 151, Val160, Gln 194, Gly 197, Leu 221, Tyr 227, Arg 229, Val 230, Ala 234, Ala282, Asp 322, Phe 327, Ala 334, Phe 338.

Thus, the invention includes mutant turkey β-adrenergic receptors inwhich, compared to its parent, one or more of these amino acid residueshave been replaced by another amino acid residue. The invention alsoincludes mutant β-adrenergic receptors from other sources in which oneor more corresponding amino acids in the parent receptor are replaced byanother amino acid residue. For the avoidance of doubt, the parent maybe a β-adrenergic receptor which has a naturally-occurring sequence, orit may be a truncated form or it may be a fusion, either to thenaturally occurring protein or to a fragment thereof, or it may containmutations compared to the naturally-occurring sequenced provided that itretains ligand-binding ability.

By “corresponding amino acid residue” we include the meaning of theamino acid residue in another β-adrenergic receptor which aligns to thegiven amino acid residue in turkey β-adrenergic receptor when the turkeyβ-adrenergic receptor and the other β-adrenergic receptor are comparedusing MacVector and CLUSTALW.

FIG. 9 shows an alignment between turkey β-adrenergic receptor and humanβ1, β2 and β3 β-adrenergic receptors.

It can be seen that Ile 72 of human β1 corresponds to Ile 55 of turkeyβ-adrenergic receptor; Ile 47 of human β2 corresponds to Ile 55 ofturkey β-adrenergic receptor; and Thr51 of human β corresponds to Ile 55of turkey β-adrenergic receptor. Other corresponding amino acid residuesin human β1, β2 and β3 can readily be identified by reference to FIG. 9.

It is preferred that the particular amino acid is replaced with an Ala.However, when the particular amino acid residue is an Ala, it ispreferred that it is replaced with a Leu (for example, see turkeyβ-adrenergic Ala 234, Ala 282 and Ala 334 in FIG. 1 ).

It is preferred if the mutant β-adrenergic receptor has a differentamino acid compared to its parent at more than one amino acid positionsince this is likely to give greater stability. Particularly preferredhuman β1 receptor mutants are those in which one or more of thefollowing amino acid residues are replaced with another amino acidresidue: K85, M107, Y244, A316, F361 and F372. Typically, the givenamino acid residue is replaced with Ala or Val or Met or Leu or Ile(unless they are already that residue).

Mutant human β1 receptors which have combinations of 3 or 4 or 5 or 6mutations as described above are prepared.

Particularly preferred human β2 receptor mutants are those in which oneor more of the following amino acids are replaced with another aminoacid residue: K60, M82, Y219, C265, L310 and F321. Typically, the givenamino acid residue is replaced with Ala or Val or Met or Leu or Ile(unless they are already that residue).

Mutant human β2 receptors which have combinations of 3 or 4 or 5 or 6mutations as described above are preferred.

FIG. 26 shows the effect on thermostability when six thermostabilisingmutations in β1-m23 (R68S, M90V, Y227A, A282L, F327A, F338M) weretransferred directly to the human β2 receptor (equivalent mutationsK60S, M82V, Y219A, C265L, L310A, F321M), making human β2-m23. The Tmsfor human β2 and β2-m23 were 29° C. and 41° C. respectively, thusexemplifying the transferability of thermostabilising mutations from onereceptor to another receptor. Accordingly, a particularly preferredhuman β2 receptor mutant is one which comprises the mutations K60S,M82V, Y219A, C265L, L310A, F321M.

Particularly preferred human β3 receptor mutants are those in which oneor more of the following amino acids are replaced with another aminoacid residue: W64, M86, Y224, P284, A330 and F341. Typically, the givenamino acid residue is replaced with Ala or Val or Met or Leu or Ile(unless they are already that residue).

Mutant human β3 receptors which have combinations of 3 or 4 or 5 or 6mutations as described above are preferred.

Particularly preferred combinations of mutations are described in detailin Tables 1 and 2 in Example 1, and the invention includes the mutantturkey β-adrenergic receptors, and also includes mutant β-adrenergicreceptors where amino acids in corresponding position have been replacedby another amino acid, typically the same amino acid as indicated inTables 1 and 2 in Example 1.

Particularly preferred mutants are those which contain mutations in theamino acids which correspond to the given amino acid residue byreference to turkey β-adrenergic receptor: (R68S, Y227A, A282L, A334L)(see m6-10 in Table 2 below); (M90V, Y227A, F338M) (see m7-7 in Table 2below); (R68S, M90V, V230A, F327A, A334L) (see m10-8 in Table 2 below);and (R68S, M90V, Y227A, A282L, F327A, F338M) (see m23 in Table 2 below).

Mutant Adenosine Receptor

Adenosine receptors are well known in the art. They share sequencehomology to each other and bind to adenosine.

A fifth aspect of the invention provides a mutant adenosine receptorwhich, when compared to the corresponding wild-type adenosine, has adifferent amino acid at a position which corresponds to any one or moreof the following positions according to the numbering of the humanadenosine A_(2a) receptor as set out in FIG. 10 : Gly 114, Gly 118, Leu167, Ala 184, Arg 199, Ala 203, Leu 208, Gln 210, Ser 213, Glu 219, Arg220, Ser 223, Thr 224, Gin 226, Lys 227, His 230, Leu 241, Pro 260, Ser263, Leu 267, Leu 272, Thr 279, Asn 284, Gln 311, Pro 313, Lys 315, Ala54, Val 57, His 75, Thr 88, Gly 114, Gly 118, Thr 119, Lys 122, Gly 123,Pro 149, Glu 151, Gly 152, Ala 203, Ala 204, Ala 231, Leu 235, Val 239.

The mutant adenosine receptor may be a mutant of any adenosine receptorprovided that it is mutated at one or more of the amino acid positionsas stated by reference to the given human adenosine A_(2a) receptoramino acid sequence.

It is particularly preferred if the mutant GPCR is one which has atleast 20% amino acid sequence identity when compared to the given humanadenosine A_(2a) receptor sequence, as determined using MacVector andCLUSTALW. Preferably, the mutant GPCR has at least 30% or at least 40%or at least 50% or at least 60% sequence identity. Typically, there is ahigher degree of sequence conservation at the adenosine binding site.

As is described in Example 2 below, individual replacement of thefollowing amino acid residues in the human adenosine A_(2a) receptorsequence (as shown in FIG. 10 ) lead to an increase in thermostabilitywhen measured with the agonist 5′-N-ethylcarboxamidoadenosine (NECA):

Gly 114, Gly 118, Leu 167, Ala 184, Arg 199, Ala 203, Leu 208, Gln 210,Ser 213, Glu 219, Arg 220, Ser 223, Thr 224, Gln 226, Lys 227, His 230,Leu 241, Pro 260, Ser 263, Leu 267, Leu 272, Thr 279, Asn 284, Gln 311,Pro 313, Lys 315.

Replacement of the following amino acid residues in the human A_(2a)receptor sequence (as shown in FIG. 10 ) lead to an increase inthermostability when measured with the antagonist ZM 241385(4-[2-[[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-α][1,3,5[triazin-5-yl]amino]ethyl]phenol):

Ala 54, Val 57, His 75, Thr 88, Gly 114, Gly 118, Thr 119, Lys 122, Gly123, Pro 149, Glu 151, Gly 152, Ala 203, Ala 204, Ala 231, Leu 235, Val239.

Thus, the invention includes mutant human adenosine A_(2a) receptors inwhich, compared to its parent, one or more of these amino acid residueshave been replaced by another amino acid residue. The invention alsoincludes mutant adenosine receptors from other sources in which one ormore corresponding amino acids in the parent receptor are replaced byanother amino acid residue. For the avoidance of doubt, the parent maybe an adenosine receptor which has a naturally-occurring sequence, or itmay be a truncated form or it may be a fusion, either to thenaturally-occurring protein or to a fragment thereof, or it may containmutations compared to the naturally-occurring sequence, provided that itretains ligand-binding ability.

By “corresponding amino acid residue” we include the meaning of theamino acid residue in another adenosine receptor which aligns to thegiven amino acid residue in human adenosine A_(2a) receptor when thehuman adenosine A_(2a) receptor and the other adenosine receptor arecompared using MacVector and CLUSTALW.

FIG. 10 shows an alignment between human adenosine A_(2a) receptor andthree other human adenosine receptors (A2b, A3 and A1).

It can be seen that, for example, Ser 115 in the A_(2b) receptor(indicated as AA2BR) corresponds to Gly 114 in the A_(2a) receptor.Similarly, it can be seen that Ala 60 in the A₃ receptor (indicated asAA3R) corresponds to Ala 54 in the A_(2a) receptor, and so on. Othercorresponding amino acid residues in human adenosine receptors A_(2b),A₃ and A₁ can readily be identified by reference to FIG. 10 .

It is preferred that the particular amino acid in the parent is replacedwith an Ala. However, when the particular amino acid residue in theparent is an Ala, it is preferred that it is replaced with a Leu.

It is preferred that the mutant adenosine receptor has a different aminoacid compared to its parent at more than one amino acid position.Particularly preferred human adenosine A2b receptors are those in whichone or more of the following amino acid residues are replaced withanother amino acid residue: A55, T89, R123, L236 and V240. Typically,the given amino acid residue is replaced with Ala or Val or Met or Leuor Ile (unless they are already that residue).

Mutant human adenosine A2b receptors which have combinations of 3 or 4or 5 mutations as described above are preferred.

Particularly preferred human adenosine A3 receptors are those in whichone or more of the following amino acid residues are replaced withanother amino acid residue: A60, T94, W128, L232 and L236. Typically,the given amino acid residue is replaced with Ala or Val or Met or Leuor Ile (unless they are already that residue).

Mutant human adenosine A3 receptors which have combinations of 3 or 4 or5 mutations as described above are preferred.

Particular preferred human adenosine A1 receptors are those in which oneor more of the following residues are replaced: A57, T91, A125, L236,and L240. Typically, the given amino acid residue is replaced with Alaor Val or Met or Leu or Ile (unless they are already that residue).

Particularly preferred combinations of mutations are described in detailin Example 2. The invention includes these mutant human adenosine A_(2a)receptors, and also includes other mutant adenosine receptors whereamino acids in corresponding positions have been replaced by anotheramino acid, typically the same amino acid as indicated in Example 2.

Particularly preferred adenosine receptor mutants are those whichcontain mutations in the amino acids which correspond to the given aminoresidue by reference to human adenosine A2a receptor: (A54L, K122A,L235A) (Rant 17); (A54L, T88A, V239A, A204L) (Rant 19); and (A54L, T88A,V239A, K122A) (Rant 21).

Mutant Neurotensin Receptor

Neurotensin receptors are known in the art. They share sequence homologyand bind neurotensin.

A sixth aspect of the invention provides a mutant neurotensin receptorwhich, when compared to the corresponding wild-type neurotensinreceptor, has a different amino acid at a position which corresponds toany one or more of the following positions according to the numbering ofthe rat neurotensin receptor as set out in FIG. 11 : Ala 69, Leu 72, Ala73, Ala 86, Ala 90, Ser 100, His 103, Ser 108, Leu 109, Leu 111, Asp113, Ile 116, Ala 120, Asp 139, Phe 147, Ala 155, Val 165, Glu 166, Lys176, Ala 177, Thr 179, Met 181, Ser 182, Arg 183, Phe 189, Leu 205, Thr207, Gly 209, Gly 215, Val 229, Met 250, Ile 253, Leu 256, Ile 260, Asn262, Val 268, Asn 270, Thr 279, Met 293, Thr 294, Gly 306, Leu 308, Val309, Leu 310, Val 313, Phe 342, Asp 345, Tyr 349, Tyr 351, Ala 356, Phe358, Val 360, Ser 362, Asn 370, Ser 373, Phe 380, Ala 385, Cys 386, Pro389, Gly 390, Trp 391, Arg 392, His 393, Arg 395, Lys 397, Pro 399.

It is particularly preferred if the mutant GPCR is one which has atleast 20% amino acid sequence identity when compared to the given ratneurotensin receptor sequence, as determined using Mac Vector andCLUSTALW. Preferably, the mutant GPCR has at least 30% or at least 40%or at least 50% amino acid sequence identity.

The mutant neurotensin receptor may be a mutant of any neurotensinreceptor provided that it is mutated at one or more of the amino acidpositions as stated by reference to the given rat neurotensin receptoramino acid sequence.

As is described in Example 3 below, individual replacement of thefollowing amino acid residues in the rat neurotensin receptor sequence(as shown in FIGS. 11 and 28 ) lead to an increase in thermostabilitywhen considered with respect to the absence of neurotensin. Leu 72, Ala86, Ala 90, Ser 100, His 103, Ser 108, Leu 109, Leu 111, Asp 113, Ile116, Ala 120, Asp 139, Phe 147, Ala 155, Lys 176, Thr 179, Met 181, Ser182, Phe 189, Leu 205, Thr 207, Gly 209, Gly 215, Leu 256, Asn 262, Val268, Met 293, Asp 345, Tyr 349, Tyr 351, Ala 356, Phe 358, Ser 362, Ala385, Cys 386, Trp 391, Arg 392, His 393, Lys 397, Pro 399.

As is described in Example 3 below, individual replacement of thefollowing amino acid residues in the rat neurotensin receptor sequence(as shown in FIGS. 11 and 28 ) lead to an increase in thermostabilitywhen considered with respect to the presence of neurotensin. Ala 69, Ala73, Ala 86, Ala 90, His 103, Val 165, Glu 166, Ala 177, Arg 183, Gly215, Val 229, Met 250, Ile 253, Ile 260, Thr 279, Thr 294, Gly 306, Leu308, Val 309, Leu 310, Val 313, Phe 342, Phe 358, Val 360, Ser 362, Asn370, Ser 373, Phe 380, Ala 385, Pro 389, Gly 390, Arg 395.

Thus, the invention includes mutant rat neurotensin receptor in which,compared to its parent, one or more of these amino acid residues havebeen replaced by another amino acid residue. The invention also includesmutant neurotensin receptors from other sources in which one or morecorresponding amino acids in the parent receptor are replaced by anotheramino acid residue. For the avoidance of doubt the parent may be aneurotensin receptor which has a naturally-occurring sequence, or it maybe a truncated form or it may be a fusion, either to thenaturally-occurring protein or to a fragment thereof, or it may containmutations compared to the naturally-occurring sequence, providing thatit retains ligand-binding ability.

By “corresponding amino acid residue” we include the meaning of theamino acid residue in another neurotensin receptor which aligns to thegiven amino acid residue in rat neurotensin receptor when the ratneurotensin receptor and the other neurotensin receptor are comparedusing MacVector and CLUSTALW.

FIG. 11 shows an alignment between rat neurotensin receptor and twohuman neurotensin receptors 1 and 2. It can be seen, for example, thatAla 85 of the human neurotensin receptor 1 corresponds to Ala 86 of therat neurotensin receptor, that Phe 353 of the human neurotensin receptor1 corresponds to Phe 358 of the rat neurotensin receptor, and so on.Other corresponding amino acid residue in the human neurotensinreceptors 1 and 2 can readily be identified by reference to FIG. 11 .

It is preferred that the particular amino acid in the parent is replacedwith an Ala. However, when the particular amino acid residue in theparent is an Ala, it is preferred that it is replaced with a Leu.

It is preferred that the mutant neurotensin receptor has a differentamino acid compared to its parent at more than one amino acid position.Particularly preferred human neurotensin receptors (NTR1) are those inwhich one or more of the following amino acid residues are replaced withanother amino acid residue: Ala 85, His 102, Ile 259, Phe 337 and Phe353. Typically, the given amino acid residues is replaced with Ala orVal or Met or Leu or Ile (unless they are already that residue).

Mutant human neurotensin receptors (NTR1) which have combinations of 3or 4 or 5 mutations as described above are preferred.

Particularly preferred human neurotensin receptors (NTR2) are those inwhich one or more of the following amino acid residues are replaced withanother amino acid residue: V54, R69, T229, P331 and F347. Typically,the given amino acid residue is replaced with Ala or Val or Met or Leuor Ile (unless they are already that residue). Mutant human neurotensinreceptors (NTR2) which have combinations of 3 or 4 or 5 mutations asdescribed above are preferred.

Particularly preferred combinations of mutations are described in detailin Example 3. The invention includes these mutant rat neurotensinreceptors, and also includes other mutant neurotensin receptors whereamino acids in corresponding positions have been replaced by anotheramino acid, typically the same amino acid as indicated in Example 3.

Particularly preferred neurotensin receptor mutants are those whichcontain mutations in the amino acid residues which correspond to thegiven amino acid residue by reference to the rat neurotensin receptor:(F358A, A86L, 1260A, F342A) (Nag7m); (F358A, H103A, 1260A, F342A)(Nag7n).

Mutant Muscarinic Receptor

Muscarinic receptors are known in the art. They share sequence homologyand bind muscarine.

A seventh aspect of the invention provides a mutant muscarinic receptorwhich, when compared to the corresponding wild-type muscarinic receptor,has a different amino acid at a position which corresponds to any one ormore of the following positions according to the numbering of the humanmuscarinic receptor M1 as set out in FIG. 17 : Leu 65, Met 145, Leu 399,Ile 383 and Met 384.

It is particularly preferred if the mutant GPCR is one which has atleast 20% amino acid sequence identity when compared to the given humanmuscarinic receptor sequence, as determined using MacVector andCLUSTALW. Preferably, the mutant GPCR has at least 30% or at least 40%or at least 50% amino acid sequence identity.

The mutant muscarinic receptor may be a mutant of any muscarinicreceptor provided that it is mutated at one or more of the amino acidpositions as stated by reference to the given muscarinic receptor aminoacid sequence.

Thus, the invention includes a mutant human muscarinic receptor inwhich, compared to its parent, one or more of these amino acid residueshave been replaced by another amino acid residue. The invention alsoincludes mutant muscarinic receptors from other sources in which one ormore corresponding amino acids in the parent receptor are replaced byanother amino acid residue. For the avoidance of doubt the parent may bea muscarinic receptor which has a naturally-occurring sequence, or itmay be a truncated form or it may be a fusion, either to thenaturally-occurring protein or to a fragment thereof, or it may containmutations compared to the naturally-occurring sequence, providing thatit retains ligand-binding ability.

By “corresponding amino acid residue” we include the meaning of theamino acid residue in another muscarinic receptor which aligns to thegiven amino acid residue in human muscarinic receptor when the humanmuscarinic receptor and the other muscarinic receptor are compared usingMacVector and CLUSTALW.

It is preferred that the particular amino acid is replaced with an Ala.However, when the particular amino acid residue is an Ala, it ispreferred that it is replaced with a Leu.

As shown in Examples 1-3 and described above, we have identifiedthermostabilising mutations scattered widely throughout the sequences ofthe turkey beta1 adrenergic receptor, human adenosine receptor, ratneurotensin receptor and human muscarinic receptor. FIG. 17 provides analignment of these sequences with the sequence of the human beta-2ARsuch that when the thermostabilising mutations are positioned onto thesequences then, in 11 instances out of a total of 70, two sequencescontain mutations at the same position (denoted in FIG. 17 with a star).Thus it will be appreciated that once one or more stabilising mutationshave been identified in one GPCR, a further GPCR with increasedstability can be generated by aligning the amino acid sequences of theGPCRs and making the same one or more mutations at the correspondingposition or positions. This concept is clearly exemplified in FIG. 26wherein the six thermostabilising mutations in turkey β1-m23 weretransferred directly to the human β2 receptor. The resultant mutant,β2-m23, had a Tm 12° C. higher than that of the human β2 receptor.

Accordingly, an eighth aspect of the invention provides a method forproducing a mutant GPCR with increased stability relative to its parentGPCR, the method comprising:

-   -   (i) identifying in the amino acid sequence of one or more        mutants of a first parent GPCR with increased stability relative        to the first parent GPCR, the position or positions at which the        one or more mutants have at least one different amino acid        residue compared to the first parent GPCR, and    -   (ii) making one or more mutations in the amino acid sequence        that defines a second GPCR at the corresponding position or        positions, to provide one or more mutants of a second parent        GPCR with increased stability relative to the second parent        GPCR.

The one or more mutants of a first parent GPCR may be selected orprepared according to the methods of the first or second aspects of theinvention. Accordingly, it will be appreciated that the one or moremutants of a first parent GPCR may be any of the mutants of the third,fourth, fifth, sixth or seventh aspects of the invention. Hence, themethod of the eighth aspect of the invention may be used to createstable, conformationally locked GPCRs by mutagenesis. For example,following the selection of mutant GPCRs which have increased stabilityin a particular conformation, the stabilising mutation can be identifiedand the amino acid at a corresponding position in a second GPCR replacedto produce a mutant GPCR with increased stability in a particularconformation relative to a second parent GPCR.

For the avoidance of doubt the first parent GPCR may be a GPCR which hasa naturally-occurring sequence, or it may be a truncated form or it maybe a fusion, either to the naturally-occurring protein or to a fragmentthereof, or it may contain mutations compared to the naturally-occurringsequence, providing that it retains ligand-binding ability.

Typically, identifying the position or positions at which the one ormore mutants have at least one different amino acid residue compared tothe first parent GPCR involves aligning their amino acid sequences withthat of the parent GPCR, for example using the Clustal W program(Thompson et al., 1994).

By “corresponding position or positions”, we include the meaning of theposition in the amino acid sequence of a second GPCR which aligns to theposition in the amino acid sequence of the first GPCR, when the firstand second GPCRs are compared by alignment, for example by usingMacVector and Clustal W. For example, as shown in the alignment in FIG.17 , the six stabilising mutations in turkey β1-m23, R68S, M90V, Y227A,A282L, F327A and F338M, are at positions which correspond to residuesK60, M82, Y219, C265, L310 and F321 respectively in the human β2receptor.

Having identified the corresponding position or positions in the aminoacid sequence of a second GPCR, the amino acids at those positions arereplaced with another amino acid. Typically, the amino acids arereplaced with the same amino acids which replaced the amino acids at thecorresponding positions in the mutant of the first parent GPCR (unlessthey are already that residue). For example, at position 68 in turkeyβ1-m23 (R68S), an arginine residue was replaced with a serine residue.Therefore, at the corresponding position in the human β2 receptor,position 60 (K60), the lysine residue is preferably replaced with aserine residue.

Mutations can be made in an amino acid sequence, for example, asdescribed above and using techniques well-established in the art.

It will be appreciated that the second GPCR may be any other GPCR. Forexample, stabilising mutations in a GPCR from one species may betransferred to a second GPCR from another species. Similarly,stabilising mutations in one particular GPCR isoform may be transferredto a second GPCR which is a different isoform. Preferably, the secondparent GPCR is of the same GPCR class or family as the first parentGPCR. Phylogenetic analyses have divided GPCRs into three main classesbased on protein sequence similarity, i.e., classes 1, 2, and 3 whoseprototypes are rhodopsin, the secretin receptor, and the metabotropicglutamate receptors, respectively (Foord et al (2005) Pharmacol. Rev.57, 279-288). Thus, the second GPCR may be a GPCR which is of the sameGPCR class as the first parent GPCR. Similarly, GPCRs have been dividedinto families by reference to natural ligands such as glutamate andGABA. Thus, the second GPCR may be of the same GPCR family as the firstparent GPCR. A list of GPCR classes and families has been produced bythe International Union of Pharmacology (Foord et al (2005) Pharmacol.Rev. 57, 279-288) and this list is periodically updated atwww.iuphar-db.org/GPCR/ReceptorFamiliesForward.

It will be appreciated that the second parent GPCR must be able to bealigned with the first parent GPCR such that the corresponding positionsof the mutations in the first GPCR can be determined in the second GPCR.Thus typically, the second parent GPCR has at least 20% sequenceidentity to the first parent GPCR and more preferably at least 30%, 40%,50%, 60%, 70%, 80% or 90% sequence identity to the first parent GPCR.However, some GPCRs have low sequence identity (e.g. family B and CGPCRs) and at the same time are very similar in structure. Thus the 20%sequence identity threshold is not absolute.

The inventors have reasoned that the identification of structural motifsin which the one or more mutations in a mutant GPCR with increasedstability reside, will be useful in producing further mutant GPCRs withincreased stability.

Accordingly, a ninth aspect of the invention provides a method forproducing a mutant G-protein coupled receptor (GPCR) with increasedstability relative to its parent GPCR, the method comprising:

-   -   (i) providing one or more mutants of a first parent GPCR with        increased stability relative to the first parent GPCR    -   (ii) identifying in a structural membrane protein model the        structural motif or motifs in which the one or more mutants have        at least one different amino acid residue compared to the first        parent GPCR, and    -   (iii) making one or more mutations in the amino acid sequence        that defines a corresponding structural motif or motifs in a        second parent GPCR, to provide one or more mutants of a second        parent GPCR with increased stability relative to the second        parent GPCR.

Mapping stabilising mutations onto one or more known structural modelscan be used to identify particular structural motifs in which suchstabilising mutations reside. We have mapped stabilising mutations ofthe β1-adrenergic receptor onto structural models of the β2-adrenergicreceptor (Rasmussen et al (2007) Nature 450, 383-387; Cherezov et al(2007) Science 318:1258-65; Rosenbaum et al (2007) Science318:1266-1273) in order to identify such motifs. For example, Table (vi)lists the turkey β1-adrenergic receptor mutations which we have mappedonto the human β2-adrenergic receptor and describes the correspondingstructural motifs in which they reside. As discussed in Example 4,mapping of the Y227A mutation (equivalent to Y219 in the human β₂receptor) onto the human β₂-adrenergic receptor reveals its position atthe interface between helices such that the mutation may improve packingat the helical interface (see FIGS. 15, 16 and 23 ). Similarly, mappingof the M90V mutation (equivalent to M82 in the human β₂ receptor) ontothe human β₂-adrenergic receptor reveals it to be in helix 2 at a pointwhere the helix is kinked (see FIGS. 15, 16 and 20 ). Other mutationswere found to reside in further structural motifs includingtransmembrane helix surfaces pointing into the lipid bilayer,hydrophobic-hydrophilic boundary regions, protein binding pockets andloop regions (see Table (vi) and FIGS. 18-19, 21-22 and 24-25 ).

Such structural motifs, by virtue of them containing stabilisingmutations, are important in determining protein stability. Therefore,targeting mutations to these motifs will facilitate the generation ofstabilised mutant GPCRs. Indeed, there were several instances where morethan one mutation mapped to the same structural motif. For example, theY227A, V230A and A234L mutations in the turkey β1 adrenergic receptormapped to the same helical interface, the V89L and M90V mutations mappedto the same helical kink and the F327A and A334L mutations mapped to thesame helical surface pointing towards the lipid bilayer (Table (vi)).Thus, when one stabilising mutation has been identified, thedetermination of the structural motif in which that mutation is locatedwill enable the identification of further stabilising mutations.

In an embodiment of the ninth aspect of the invention, the one or moremutants of a first parent GPCR are selected or prepared according to themethods of the first, second or eighth aspects of the invention.Accordingly, it will be appreciated that the one or more mutants of afirst parent GPCR may be any of the mutants of the third, fourth, fifth,sixth or seventh aspects of the invention. Hence, the method of theninth aspect of the invention may also be used to create stable,conformationally locked GPCRs by mutagenesis. For example, following theselection of mutant GPCRs which have increased stability in a particularconformation, the structural motifs in which such stabilising mutationsreside can be identified. Making one or more mutations in the amino acidsequence that defines the corresponding structural motif in another GPCRcan then be used to produce a mutant GPCR with increased stability in aparticular conformation relative to its parent GPCR.

We have performed a multiple sequence alignment of the human beta-2AR,rat NTR1, turkey beta-1 AR, human Adenosine A2aR and human muscarinic M1receptor amino acid sequences (FIG. 17 ) which shows that, when thethermostabilising mutations identified (see Examples 1-3) are positionedon the sequences then, in 11 instances out of a total of 70, twosequences contain mutations at the same position (denoted in FIG. 17with a star). Without wishing to be bound by any theory, the inventorsbelieve that thermostabilising mutations at these positions should be ofenhanced transferability for mapping onto a structural membrane proteinmodel. Thus in one embodiment, the mutant of the first parent GPCR is amutant human beta-2AR, rat NTR1, turkey beta-1 AR, human Adenosine A2aRor human muscarinic M1 receptor which, when compared to itscorresponding parent receptor, has a different amino acid at a positionwhich corresponds to any one or more of the following positionsaccording to the numbering of the human beta2 AR as set out in FIG. 17 :Ala 59, Val 81, Ser 143, Lys 147, Val 152, Glu 180, Val 222, Ala 226,Ala 271, Leu 275 and Val 317.

In order to identify the structural motif or motifs, the stabilisingmutations are mapped onto a known structure of a membrane protein.

By “membrane protein” we mean a protein that is attached to orassociated with a membrane of a cell or organelle. Preferably, themembrane protein is an integral membrane protein that is permanentlyintegrated into the membrane and can only be removed using detergents,non-polar solvents or denaturing agents that physically disrupt thelipid bilayer.

The structural model of a membrane protein may be any suitablestructural model. For example, the model may be a known crystalstructure. Examples of GPCR crystal structures include bovine rhodopsin(Palczewski, K. et al., Science 289, 739-745. (2000)) and human β₂adrenergic receptor (Rasmussen et al, Nature 450, 383-7 (2007); Cherezovet al (2007) Science 318:1258-65; Rosenbaum et al (2007) Science318:1266-1273). The coordinates for the human β₂ adrenergic receptorstructure can be found in the RCSB Protein Data Bank under accessioncodes: 2rh1, 2r4r and 2r4s. Alternatively, the structural model may be acomputer generated model based upon homology or using de novo structureprediction methods (Qian et al Nature (2007) 450: 259-64).

It will be appreciated that stabilising mutations of a given mutant GPCRcan be mapped onto a structural model of any membrane protein which hassufficient structural similarity to the GPCR. In particular, the domainof the membrane protein must have sufficient structural similarity tothe GPCR domain in which the stabilising mutation resides, for a givenmutation to be transferable.

A protein domain is typically defined as a discretely folded assembly ofsecondary structure elements which may stand alone as a single proteinor be part of a larger protein in combination with other domains. It iscommonly a functional evolutionary unit.

GPCRs are essentially single domain proteins excluding those with largeN-terminal domains. Therefore, typically, the structural model is of amembrane protein which comprises at least one domain that has sufficientstructural similarity to the GPCR.

Structural similarity can be determined indirectly by the analysis ofsequence identity, or directly by comparison of structures.

With regard to sequence identity, the amino acid sequence encoding theGPCR domain in which the mutant has at least one different amino acidresidue compared to the first parent GPCR, is aligned with an amino acidsequence encoding a domain of a membrane protein for which a structuralmodel is available. It will be appreciated that one or more of thesesequences may contain an inserted sequence or N-terminal or C-terminalextensions which are additional to the core conserved domain. Foroptimal alignment, such sequences are removed so as not to skew theanalysis. Membrane proteins with sufficient sequence identity across thedomain in question may then be used as the structural model for mappingmutations. It has been shown for soluble protein domains that their 3Dstructure is broadly conserved above 20% sequence identity and wellconserved above 30% identity, with the level of structural conservationincreasing as sequence identity increases up to 100% (Ginalski, K. CurrOp Struc Biol (2006) 16, 172-177). Thus, it is preferred if thestructural membrane protein model is a model of a membrane protein whichcontains a domain that shares at least 20% sequence identity with themutant GPCR domain containing the at least one different amino acidresidue compared to the first parent GPCR, and more preferably at least30%, 40%, 50%, 60%, 70%, 80% or 90% sequence identity, and yet morepreferably at least 95% or 99% sequence identity.

Sequence identity may be measured by the use of algorithms such as BLASTor PSI-BLAST (Altschul et al, NAR (1997), 25, 3389-3402) or methodsbased on Hidden Markov Models (Eddy S et al, J Comput Biol (1995) Spring2 (1) 9-23). Typically, the percent sequence identity between twopolypeptides may be determined using any suitable computer program, forexample the GAP program of the University of Wisconsin Genetic ComputingGroup and it will be appreciated that percent identity is calculated inrelation to polypeptides whose sequence has been aligned optimally. Thealignment may alternatively be carried out using the Clustal W program(Thompson et al., 1994). The parameters used may be as follows: Fastpairwise alignment parameters: K-tuple (word) size; 1, window size; 5,gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.Multiple alignment parameters: gap open penalty; 10, gap extensionpenalty; 0.05. Scoring matrix: BLOSUM.

In addition to sequence identity, structural similarity can bedetermined directly by comparison of structural models. Structuralmodels may be used to detect regions of structural similarity notevident from sequence analysis alone, and which may or may not becontiguous in the sequence. For example, family B and C GPCRs arethought to share similar structures; however, their sequence identity isvery low. Similarly, the water transporting aquaporins spinach SoPip2,E. coli AqpZ, Methanococcus AqpM, rat Aqp4, human Aqp1 and sheep Aqp0share low sequence identity but all have similar structures.

Structural models of high fidelity may be constructed for proteins ofunknown structure using standard software packages such as MODELLER(Sali A and Blundell T, J Mol Biol (1993) 234(3) 779-815), wherein thestructure is modelled on a known structure of a homologous protein. Suchmodelling improves with increasing sequence identity. Typically, thesequence identity between the sequence of unknown structure and asequence of known 3D structure is more than 30% (Ginalski, K. Curr OpStruc Biol (2006) 16, 172-177). In addition, de novo structureprediction methods based on sequence alone may be used to model proteinsof unknown structure (Qian et al, (2007) Nature 450:259-64). Oncestructures have been experimentally determined or derived by modelling,regions of structural similarity may be detected by direct comparison oftwo or more 3D structures. They may, for example, comprise secondarystructure elements of a particular architecture and topology which canbe detected by the use of software such as DALI (Holm, L and Sander, C(1996) Science 273, 595-603). They may comprise local arrangements ofamino acid side chains and the polypeptide backbone, or specific sets ofatoms or groups of atoms in a particular spatial arrangement, which mayfor example also be detected by the use of graph theoreticalrepresentations (Artymiuk,P et al, (2005) J Amer Soc Info Sci Tech 56(5) 518-528). In this approach, the atoms or groups of atoms within theproteins or regions of proteins to be compared are typically representedas the nodes of a graph, with the edges of the graph describing theangles and distances between the nodes. Common patterns in these graphsindicate common structural motifs. This approach may be extended toinclude any descriptor of atoms or groups of atoms, such as hydrogenbond donor or acceptor, hydrophobicity, shape, charge or aromaticity;for example proteins may be spatially mapped according to suchdescriptors using GRID and this representation used as a basis forsimilarity searching (Baroni et al (2007) J Chem Inf Mod 47, 279-294).Descriptions of the methods, availability of software, and guidelinesfor user-defined selection of parameters, thresholds and tolerances aredescribed in the references given above.

In a preferred embodiment, the structural membrane protein model is astructural GPCR model. It will be appreciated that the structural modelof a GPCR may be a model of the first parent GPCR. For example,stabilising mutations within a mutant GPCR having increased stabilitycan be directly mapped onto the first parent GPCR structure and thestructural motifs in which such mutations are located, identified. Wherethe structure of the first parent GPCR is unknown, structural models ofother GPCRs may be used. For example, stabilising mutations in a GPCRfrom one species may be mapped onto a known structural model of the sameGPCR from another species. Similarly, stabilising mutations in oneparticular GPCR isoform may be mapped onto a known structural model ofanother GPCR isoform. Moreover, stabilising mutations from one GPCR maybe mapped onto a GPCR of the same class or family. A list of GPCRclasses and families has been produced by the International Union ofPharmacology (Foord et al (2005) Pharmacol. Rev. 57, 279-288) and thislist is periodically updated atwww.iuphar-db.org/GPCR/ReceptorFamiliesForward.

As described above, it will be appreciated that the structural model maybe of any GPCR provided it has sufficient structural similarity acrossthe domain in which the mutant GPCR has at least one different aminoacid compared to the first parent GPCR. Thus, it is preferred if theGPCR shares at least 20% sequence identity with the mutant of the firstparent GPCR across the protein domain containing the at least onedifferent amino acid residue compared to the first parent GPCR, and morepreferably at least 30%, 40%, 50%, 60%, 70%, 80% or 90% sequenceidentity, and yet more preferably at least 95% or 99% sequence identity.However, the inventors recognise that the 20% sequence identitythreshold is not absolute. GPCRs with less than 20% sequence identity tothe first parent GPCR may also serve as a structural model to whichstabilising mutations are transferred, wherein the low sequence identityis counterbalanced by other similarities, including, for example, thepresence of the same sequence motifs, binding to the same G-protein orhaving the same function, or having substantially the same hydropathyplots compared to the first parent GPCR.

Mapping of stabilising mutations onto the structural model can be doneusing any suitable method known in the art. For example, typically, theamino acid sequence of the GPCR for which the structural model isavailable is aligned with the amino acid sequence of the mutant of thefirst parent GPCR. The position or positions of the at least onedifferent amino acid residue in the mutant GPCR relative to the firstparent GPCR can then be located in the amino acid sequence of the GPCRfor which a structural model is available.

By ‘structural motif’ we include the meaning of a three dimensionaldescription of the location in a GPCR structural model of athermostabilising mutation. For example, the structural motif may be anysecondary or tertiary structural motif within the GPCR. By ‘tertiarystructural motif’ we include any descriptor of atoms or groups of atoms,such as hydrogen bond donor or acceptor, hydrophobicity, shape, chargeor aromaticity. For example, proteins may be spatially mapped accordingto such descriptors using GRID and this representation used as a basisfor defining a structural motif (Baroni et al (2007) J Chem Inf Mod 47,279-294).

Table (vi) lists the structural motifs in which the turkey β1 adrenergicreceptor stabilising mutations were found to reside. As seen from thetable, the mutations are positioned in a number of distinct localities.Three mutations are in loop regions that are predicted to be accessibleto aqueous solvent. Eight mutations are in the transmembrane α-helicesand point into the lipid bilayer; three of these mutations are near theend of the helices and may be considered to be at thehydrophobic-hydrophilic boundary layer. Eight mutations are found at theinterfaces between transmembrane α-helices, three of which are eitherwithin a kinked or distorted region of the helix and another twomutations occur in one helix but are adjacent to one or more otherhelices which contain a kink adjacent in space to the mutated residue.These latter mutations could affect the packing of the amino acidswithin the kinked region, which could result in thermostabilisation.Another mutation is in a substrate binding pocket.

Accordingly, in one embodiment, the structural motif is any of a helicalinterface, a Do helix kink, a helix opposite a helix kink, a helixsurface pointing into the lipid bilayer, a helix surface pointing intothe lipid bilayer at the hydrophobic-hydrophilic boundary layer, a loopregion or a protein binding pocket.

Identifying a structural motif in which a stabilising mutation residessuggests the importance of that motif in protein stability. Therefore,making one or more mutations in the amino acid sequence that defines acorresponding structural motif or motifs in a second parent GPCR, shouldprovide one or more mutants of a second parent GPCR with increasedstability relative to the second parent GPCR.

The amino acid sequence which defines a structural motif is the primaryamino acid sequence of the amino acid residues which combine in thesecondary or tertiary structure of the protein to form the structuralmotif. It will be appreciated that such a primary amino acid sequencemay comprise contiguous or non-contiguous amino acid residues. Thus,identifying the amino acid sequence which defines the structural motifwill involve determining the residues involved and subsequently definingthe sequence. Mutations can be made in an amino acid sequence, forexample as described above and using techniques well-established in theart.

By “corresponding structural motif or motifs”, we mean the analogousstructural motif or motifs identified in the structural model which arepresent in the second parent GPCR. For example, if a helical interfacewas identified, the corresponding helical interface in the second parentGPCR would be the interface between the helices which are analogous tothe helices present in the structural model. If a helical kink wasidentified, the corresponding helical kink would be the kink in thehelix which is analogous to the kinked helix present in the structuralmodel. An analogous structural motif or motifs in the second parent GPCRcan be identified by searching for similar amino acid sequences in thesequence of the second parent GPCR which define the motif or motifs inthe structural model, for example, by sequence alignment. Moreover,computer based algorithms are widely available in the art that can beused to predict the presence of protein motifs based on an amino acidsequence. Thus, based upon the relative position of a particular motifwithin the amino acid sequence and its position relative to othermotifs, an analogous structural motif can readily be identified. It willbe appreciated that if a structural model of the second parent GPCR isavailable, the analogous structural motif or motifs can be directlymapped onto the structure of the protein. Typically, the amino acidsequence defining the analogous structural motif has at least 20%sequence identity with the sequence defining the motif in the structuralmodel, more preferably at least 30%, 40%, 50%, 60%, 70%, 80% and 90%sequence identity and yet more preferably 95% and 99% sequence identity.

In one embodiment, the second parent GPCR is the first parent GPCR. Forthe avoidance of doubt, the second parent GPCR may have thenaturally-occurring sequence of the first parent GPCR, or it may be atruncated form or it may be a fusion, either to the naturally occurringprotein or to a fragment thereof, or it may contain mutations comparedto the naturally-occurring sequence, providing that it retainsligand-binding.

In an alternative embodiment, the second parent GPCR is not the firstparent GPCR. For example, a mutant of a first parent GPCR may have beenidentified that has increased stability but it is desired to generate amutant of a different GPCR with increased stability. Preferably, thesecond parent GPCR is of the same GPCR class or family as the firstparent GPCR as described above. However, it will be appreciated that thesecond parent GPCR may be any known GPCR provided that it sharessufficient structural similarity with the first parent GPCR, such thatit contains a corresponding structural motif in which the stabilisingmutation of the mutant of the first parent GPCR resides. Thus typically,the second parent GPCR has at least 20% sequence identity to the firstparent GPCR and more preferably at least 30%, 40%, 50%, 60%, 70%, 80% or90% sequence identity. However, as mentioned above, some GPCRs have lowsequence identity (e.g. family B and C GPCRs) but are similar instructure. Thus the 20% sequence identity threshold is not absolute.

Since there are potentially thousands of mutations that can be screenedin a GPCR for increased stability, it is advantageous to targetparticular mutations which are known to be important in conferringstability. Therefore, it will be appreciated that the methods of theeighth and ninth aspects of the invention may be used in a method ofselecting mutant GPCRs with increased stability. In particular, carryingout the methods of the eighth or ninth aspects of the invention can beused to target mutations to particular amino acid residues or to aminoacid sequences which define structural motifs important in determiningstability.

Accordingly, in one embodiment the methods of the eighth or ninthaspects further comprise:

-   -   (I) selecting a ligand, the ligand being one which binds to the        second parent GPCR when the GPCR is residing in a particular        conformation    -   (II) determining whether the or each mutant of the second parent        GPCR when residing in a particular conformation has increased        stability with respect to binding the selected ligand compared        to the stability of the second parent GPCR when residing in the        same particular conformation with respect to binding that        ligand, and    -   (III) selecting those mutants that have an increased stability        compared to the second parent GPCR with respect to binding the        selected ligand.

It will be noted that steps (I), (II) and (III) correspond to steps (b),(c) and (d) of the method of the first aspect of the invention describedabove. Accordingly, preferences for the ligand and methods of assessingstability are as defined above with respect to the method of the firstaspect of the invention.

A tenth aspect of the invention provides a mutant GPCR with increasedstability relative to its parent GPCR produced by the method of thetenth aspect of the invention.

In one embodiment, the mutant GPCR of the tenth aspect of the inventionis a mutant GPCR which has, compared to its parent receptor, at leastone different amino acid at a position which corresponds to any one ormore of the following positions: (i) according to the numbering of theturkey β-adrenergic receptor as set out in FIG. 9 : Ile 55, Gly 67, Arg68, Val 89, Met 90, Gly 67, Ala 184, Arg 199, Ala 203, Leu 208, Gln 210,Ser 213, Glu 219, Arg 220, Ser 223, Thr 224, Gln 226, Lys 227, His 230,Leu 241, Pro 260, Ser 263, Leu 267, Leu 272, Thr 279, Asn 284, Gln 311,Pro 313, Lys 315, (iii) according to the numbering of the ratneurotensin receptor as set out in FIG. 11 : Ala 69, Leu 72, Ala 73, Ala86, Ala 90, Ser 100, His 103, Ser 108, Leu 109, Leu 111, Asp 113, Ile116, Ala 120, Asp 139, Phe 147, Ala 155, Val 165, Glu 166, Lys 176, Ala177, Thr 179, Met 181, Ser 182, Arg 183, Phe 189, Leu 205, Thr 207, Gly209, Gly 215, Val 229, Met 250, Ile 253, Leu 256, Ile 260, Asn 262, Val268, Asn 270, Thr 279, Met 293, Thr 294, Gly 306, Leu 308, Val 309, Leu310, Val 313, Phe 342, Asp 345, Tyr 349, Tyr 351, Ala 356, Phe 358, Val360, Ser 362, Asn 370, Ser 373, Phe 380, Ala 385, Cys 386, Pro 389, Gly390, Trp 391, Arg 392, His 393, Arg 395, Lys 397, Pro 399, and (iv)according to the numbering of the muscarinic receptor as set out in FIG.17 : Leu 65, Met 145, Leu 399, Ile 383 and Met 384.

Alignment of the turkey β1 AR, human adenosine receptor, rat neurotensinreceptor and human muscarinic receptor amino acid sequences in FIG. 17 ,shows that in 11 instances out of 70, two sequences contain mutationsart the same position, namely at the following positions according tothe numbering of the human beta2 AR as set out in FIG. 17 : Ala 59, Val81, Ser 143, Lys 147, Val 152, Glu 180, Val 222, Ala 226, Ala 271, Leu275 and Val 317. Therefore, in a preferred embodiment, the mutant GPCRof the tenth aspect of the invention is one which has, compared to itsparent receptor, a different amino acid at any one of these positions.

In one embodiment the mutant GPCR of the tenth aspect of the inventionis a mutant β-adrenergic receptor. For example, the mutant β-adrenergicreceptor may have at least one different amino acid residue in astructural motif in which the mutant receptor compared to its parentreceptor has a different amino acid at a position which corresponds toany of the following positions according to the numbering of the turkeyβ-adrenergic receptor as set out in FIG. 9 : Ile 55, Gly 67, Arg 68, Val89, Met 90, Gly 98, Ile 129, Ser 151, Val 160, Gln 194, Gly 197, Leu221, Tyr 227, Arg 229, Val 230, Ala 234, Ala 282, Asp 322, Phe 327, Ala334, Phe 338.

In one embodiment the mutant GPCR of the tenth aspect of the inventionis a mutant adenosine receptor. For example, the mutant adenosinereceptor may have at least one different amino acid residue in astructural motif in which the mutant receptor compared to its parentreceptor has a different amino acid at a position which corresponds toany of the following positions according to the numbering of the humanadenosine Ata receptor as set out in FIG. 10 : Gly 114, Gly 118, Leu167, Ala 184, Arg 199, Ala 203, Leu 208, Gln 210, Ser 213, Glu 219, Arg220, Ser 223, Thr 224, Gln 226, Lys 227, His 230, Leu 241, Pro 260, Ser263, Leu 267, Leu 272, Thr 279, Asn 284, Gln 311, Pro 313, Lys 315.

In one embodiment the mutant GPCR of the tenth aspect of the inventionis a mutant neurotensin receptor. For example, the mutant neurotensinreceptor may have at least one different amino acid residue in astructural motif in which the mutant receptor compared to its parentreceptor has a different amino acid at a position which corresponds toany of the following positions according to the numbering of the ratneurotensin receptor as set out in FIG. 11 : Ala 69, Leu 72, Ala 73, Ala86, Ala 90, Ser 100, His 103, Ser 108, Leu 109, Leu 111, Asp 113, Ile116, Ala 120, Asp 139, Phe 147, Ala 155, Val 165, Glu 166, Lys 176, Ala177, Thr 179, Met 181, Ser 182, Arg 183, Phe 189, Leu 205, Thr 207, Gly209, Gly 215, Val 229, Met 250, Ile 253, Leu 256, Ile 260, Asn 262, Val268, Asn 270, Thr 279, Met 293, Thr 294, Gly 306, Leu 308, Val 309, Leu310, Val 313, Phe 342, Asp 345, Tyr 349, Tyr 351, Ala 356, Phe 358, Val360, Ser 362, Asn 370, Ser 373, Phe 380, Ala 385, Cys 386, Pro 389, Gly390, Trp 391, Arg 392, His 393, Arg 395, Lys 397, Pro 399.

In one embodiment the mutant GPCR of the tenth aspect of the inventionis a mutant muscarinic receptor. For example, the mutant muscarinicreceptor may have at least one different amino acid residue in astructural motif in which the mutant receptor compared to its parentreceptor has a different amino acid at a position which corresponds toany of the following positions according to the numbering of the humanmuscarinic receptor as set out in FIG. 17 : Leu 65, Met 145, Leu 399,Ile 383 and Met 384.

It is preferred that the mutant GPCRs of the invention have increasedstability to any one of heat, a detergent, a chaotropic agent and anextreme of pH.

It is preferred if the mutant GPCRs of the invention have increasedthermostability.

It is preferred that the mutant GPCRs of the invention, including themutant β-adrenergic, adenosine and neurotensin receptors, have anincreased thermostability compared to its parent when in the presence orabsence of a ligand thereto. Typically, the ligand is an antagonist, afull agonist, a partial agonist or an inverse agonist, whetherorthosteric or allosteric. As discussed above, the ligand may be apolypeptide, such as an antibody.

It is preferred that the mutant GPCRs of the invention, for example amutant β-adrenergic receptor or a mutant adenosine receptor or a mutantneurotensin receptor is at least 2° C. more stable than its parentpreferably at least 5° C. more stable, more preferably at least 8° C.more stable and even more preferably at least 10° C. or 15° C. or 20° C.more stable than its parent. Typically, thermostability of the parentand mutant receptors are measured under the same conditions. Typically,thermostability is assayed under a condition in which the GPCR residesin a particular conformation. Typically, this selected condition is thepresence of a ligand which binds the GPCR.

It is preferred that the mutant GPCRs of the invention, when solubilisedand purified in a suitable detergent has a similar thermostability tobovine rhodopsin purified in dodecyl maltoside. It is particularlypreferred that the mutant GPCR retains at least 50% of its ligandbinding activity after heating at 40° C. for 30 minutes. It is furtherpreferred that the mutant GPCR retains at least 50% of its ligandbinding activity after heating at 55° C. for 30 minutes.

The mutant GPCRs disclosed herein are useful for crystallisation studiesand are useful in drug discovery programmes. They may be used inbiophysical measurements of receptor/ligand kinetic and thermodynamicparameters eg by surface plasmon resonance or fluorescence basedtechniques. They may be used in ligand binding screens, and may becoupled to solid surfaces for use in high throughput screens or asbiosensor chips. Biosensor chips containing the mutant GPCRs may be usedto detect molecules, especially biomolecules.

The invention also includes a polynucleotide which encodes a mutant GPCRof the invention. In particular, polynucleotides are included whichencode the mutant β-adrenergic receptor or the mutant adenosinereceptors or the mutant neurotensin receptors of the invention. Thepolynucleotide may be DNA or it may be RNA. Typically, it is comprisedin a vector, such as a vector which can be used to express the saidmutant GPCR. Suitable vectors are ones which propagate in and/or allowthe expression in bacterial or mammalian or insect cells.

The invention also includes host cells, such as bacterial or eukaryoticcells, which contain a polynucleotide which encodes the mutant GPCR.Suitable cells include E. coli cells, yeast cells, mammalian cells andinsect cells.

The invention will now be described in more detail with respect to thefollowing Figures and Examples wherein:

FIG. 1 Amino acid changes in βAR that lead to thermostability. Stabilityquotient indicates the % remaining binding activity of the mutants afterheating the sample for 30 min at 32° C. All values are normalized toβAR₃₄₋₄₂₄ (50%, showed as a discontinuous line) to remove anyexperimental variability between assays. Bars show the stability foreach mutant. The letters on the x-axis indicate the amino acid presentin the mutant. The original amino acid and its position in βAR₃₄₋₄₂₄ isindicated below. Bars corresponding to the same amino acid in βAR₃₄₋₄₂₄are in the same colour with arrows indicating the best mutations. Errorswere calculated from duplicate measurements; the best mutants weresubsequently re-assayed to determine the Tm for each individual mutationand to give an accurate rank order of stability for each mutant (seeExample 1).

FIG. 2 Side chains in rhodopsin that are at equivalent positions to thethermostable mutations in βAR₃₄₋₄₂₄. The equivalent amino acid residuesin rhodopsin to the amino acid residues mutated in βAR₃₄₋₄₂₄ werelocated in the rhodopsin structure, based upon an alignment amongrhodopsin, β1 adrenergic receptor, neurotensin receptor, and adenosineA_(2a) receptor (data not shown). Side chains in the same transmembranehelix are shown as space filling models in the same colour. The name andposition of the amino acid residues are those in rhodopsin.

FIG. 3 Evolution of thermostability in βAR. Starting from βAR-m10-8,combinations of mutations were rearranged systematically to find theoptimum combination of mutations (see also Table 2).

FIG. 4 Stability of βAR-m23 and βAR₃₄₋₄₂₄ in the apo-state or containingthe bound antagonist [³H]-DHA. To determine Tm in the absence of ligand(apo-state, discontinuous lines), detergent-solubilised receptors wereincubated for 30 minutes at the temperatures indicated before carryingout the binding assay. For the Tm determination of the antagonist-boundform (continuous lines), detergent-solubilised receptors werepre-incubated with [³H]-DHA, followed by incubation at the temperaturesindicated. βAR-m23 (circles), and βAR₃₄₋₄₂₄ (squares). Data points arefrom duplicates measurements in a representative experiment.

FIGS. 5 a-c Competition binding of agonists to βAR-m23 and βAR₃₄₋₄₂₄.Binding assays were performed on receptors partially purified in DDM;βAR-m23 (triangles) and βAR₃₄₋₄₂₄ (squares). [³H]-DHA was used at aconcentration three times greater than the K_(D) of partially purifiedreceptor (see Methods). [³H]-DHA binding was competed with increasingconcentrations of the agonists, norepinephrine (FIG. 5 a ) andisoprenaline (FIG. 5 b ), or with an antagonist, alprenolol (FIG. 5 c ).Log EC₅₀ and corresponding EC₅₀ values for the different ligands werecalculated by nonlinear regression using GraphPad Prism software and theerror for log EC₅₀s were lower than 10%. The EC₅₀s for ligand binding toβAR₃₄₋₄₂₄ and βAR-m23 are: norepinephrine, βAR₃₄₋₄₂₄ 1.5 μM, βAR-m23 3.7mM; isoprenaline, βAR₃₄₋₄₂₄ 330 nM, βAR-m23 20 μM; alprenolol, βAR 78nM, βAR-m23 112 nM.

FIGS. 6 a-c Stability of βAR-m23 and βAR₃₄₋₄₂₄ in five differentdetergents. Samples of βAR₃₄₋₄₂₄ (FIG. 6 a ), and βAR-m23 (FIG. 6 b )solubilized in DDM were partially purified on Ni-NTA agarose columnsallowing the exchange into various different detergents: DDM (squares),DM (triangles), OG (inverted triangles), LDAO (diamonds) and NG(circles). βAR is so unstable in OG, NG and LDAO that it was notpossible to measure any activity after purification at 6° C. Assays werecarried out as described in the Methods and the Tm is shown at theintersection between the curves and the discontinuous line. Results arefrom duplicate measurements in a representative experiment performed inparallel. (FIG. 6 c ) Photomicrograph of a crystal of βAR-m23 mutant,which showed good order by X-ray diffraction.

FIG. 7 Curve of thermostability of βAR₃₄₋₄₂₄ (Tm). Binding assays wereperformed using [³H]-dihydroalprenolol (DHA) as radioligand as describedunder “Methods”. Samples were heated for 30 minutes at differenttemperatures before the assay. Tm represents the temperature at whichthe binding decreased to the 50%, value showed as a discontinuous line.Data points are from duplicates of one single experiment. Thisexperiment has been repeated several times with similar results.

FIGS. 8 a-b Saturation binding assays of membranes of βAR₃₄₋₄₂₄ andβAR-m23. Binding assays were performed as described in “Methods” using[³H]-dihydroalprenolol (DHA) as radioligand; βAR₃₄₋₄₂₄ (FIG. 8 a ) andβAR-m23 (FIG. 8 b ). Scatchard plots are shown as insets along with thecorresponding values for B_(max) and K_(D). Data points are fromduplicates of two independent experiments for each protein. Data wereanalyzed by nonlinear regression using Prism software (GraphPad).

FIGS. 9A-B Alignment of the turkey β-adrenergic receptor with human β1,β2 and β3 receptors.

FIGS. 10A-B Alignment of human adenosine receptors.

FIGS. 11A-B Alignment of neurotensin receptors.

FIG. 12 Flow chart showing the two different assay formats of ligand (+)and ligand (−) used to determine receptor thermostability.

FIGS. 13A-F Pharmacological profile of thermostable mutant adenosine A2areceptor, Rant21. Saturation binding of (FIG. 13A) antagonist and (FIG.13B) agonist to solubilised receptors. (FIGS. 13C-F) Inhibition of[³H]ZM241385 binding by increasing concentrations of antagonists (FIG.13C) XAC and (FIG. 13D) Theophylline, and agonists (FIG. 13E) NECA and(FIG. 13F) R-PIA; binding of [³H]ZM241385 (10 nM) in the absence ofunlabelled ligand was set to 100%. Each solubilised receptor wasincubated with ligands for one hour on ice in binding buffer (50 mM TrispH7.5 and 0.025% DDM) containing 400 mM NaCl (FIG. 13A, FIG. 13C-F).Data shown are from two independent experiments with each data pointmeasured in triplicate. K_(D) and K_(i) values are given in Table (iii).

FIGS. 14A-B Thermostable mutants show a decreased dependence on lipids(FIG. 14A) and an increased survival at higher concentration of DDM(FIG. 14B) upon heating compared to the wild-type receptor. Receptorswere solubilised in 1% DDM (diluted in 50 mM Tris pH7.5 and 400 mM NaCl)and immobilised on Ni-NTA agarose for the IMAC step. Exchange of buffercontaining the appropriate concentration of DDM and/or lipids wasperformed during washes and elution from the Ni-NTA beads.

FIG. 15 Mapping of the M90V, Y227A, A282L and F338M m23 mutations inturkey beta1 adrenergic receptor onto homologous residues (M82, Y219,C265 and A321 respectively) in the human beta2 adrenergic receptorstructure (Rasmussen et al (2007) Nature 15; 383-387; pdb accessioncodes 2R4R and 2R4S) reveals their position at a helical interface andhelical kink respectively. Amino acid residues in equivalent positionsto the thermostabilising mutations in the turkey β1 adrenergic receptorare shown as labelled space filling models.

FIG. 16 Mapping of m23 mutations in turkey beta1 adrenergic receptoronto homologous residues in the human beta2 adrenergic receptorstructure (Cherezov et al (2007) Science, 318:1258-65; pdb accessioncode 2RH1). The Cα trace of the β2AR is shown with the fusion moiety (T4lysozyme) removed. The six mutations in PAR-m23 (R68S, M90V, Y227A,A282L, F327A, F338M) are equivalent to amino acid residues K60, M82,Y219, C265, L310, F321 in the human β2AR. Lys60 is on the intracellularend of Helix 1 and points into the lipid-water interface. Met82 is nearthe middle of Helix 2 and points into the ligand binding pocket; thenearest distance between the substrate carazolol and the Met side chainis 5.7 Å. Tyr219 is towards the intracellular end of helix 5 and is atthe helix5-helix 6 interface. Cys265 is at the end of the loop regionbetween helices 5 and 6 and points away from the transmembrane regions.Leu310 and Phe321 are both in helix 7 and both point out into the lipidbilayer.

FIGS. 17A-C Multiple sequence alignment of human beta-2AR, rat NTR1,turkey beta-1 AR, human Adenosine A2aR and human muscarinic M1receptors. In each sequence, thermostabilising mutations are marked witha box. Mutations occurring in two or more sequences are denoted with astar.

FIG. 18 Mapping of turkey beta1AR mutation I55A (human beta2AR 147) ontohuman beta2AR structure (pdb accession code 2RH1). Mutation is at theinterface between 3 helices (H1, H2 kink, H7 kink). Left: side view;right: top view.

FIG. 19 Mapping of turkey beta1AR V89L mutation (human beta2AR V81) ontohuman beta2AR structure (pdb accession code 2RH1). Mutation is in thekink in helix 2. The helices are numbered and the bound antagonist isshown as a space filling model. Amino acid residues in equivalentpositions to the thermostabilising mutations in the turkey β1 adrenergicreceptor are shown as space filling models and are arrowed for clarity.Left: side view; right: top view.

FIG. 20 Mapping of turkey beta1AR M90V mutation (human beta2AR M82) ontohuman beta2AR structure (pdb accession code 2RH1). Mutation is in kinkin helix 2 oriented towards the binding pocket. The helices are numberedand the bound antagonist is shown as a space filling model. Amino acidresidues in equivalent positions to the thermostabilising mutations inthe turkey β1 adrenergic receptor are shown as space filling models andare arrowed for clarity. Left: side view; right: top view.

FIG. 21 Mapping of turkey beta1AR I129V mutation (human beta2AR I121)onto human beta2AR structure (pdb accession code 2RH1). Mutation isopposite a kink in helix 5. The helices are numbered and the boundantagonist is shown as a space filling model. Amino acid residues inequivalent positions to the thermostabilising mutations in the turkey β1adrenergic receptor are shown as space filling models and are arrowedfor clarity. Left: side view; right: bottom view.

FIG. 22 Mapping of turkey beta1AR F338M mutation (human beta2AR F321)onto human beta2AR structure (pdb accession code 2RH1). Mutation is inkink in helix 7. The helices are numbered and the bound antagonist isshown as a space filling model. Amino acid residues in equivalentpositions to the thermostabilising mutations in the turkey β1 adrenergicreceptor are shown as space filling models and are arrowed for clarity.Left: side view; right: top view.

FIG. 23 Mapping of turkey beta1AR Y227A mutation (human beta2AR Y219)onto human beta2AR structure (pdb accession code 2RH1). Mutation is athelix-helix interface. The helices are numbered and the bound antagonistis shown as a space filling model. Amino acid residues in equivalentpositions to the thermostabilising mutations in the turkey β1 adrenergicreceptor are shown as space filling models and are arrowed for clarity.Left: side view; right: bottom view.

FIG. 24 Mapping of turkey beta1AR A282L mutation (human beta2AR C265)onto human beta2AR structure (pdb accession code 2RH1). Mutation is inloop region. The helices are numbered and the bound antagonist is shownas a space filling model. Amino acid residues in equivalent positions tothe thermostabilising mutations in the turkey β1 adrenergic receptor areshown as space filling models and are arrowed for clarity. Left: sideview; right: top view.

FIG. 25 Mapping of turkey beta1 AR R68S mutation (human beta2AR K60)onto human beta2AR structure (pdb accession code 2RH1). Mutation is atthe lipid-water boundary, pointing into the solvent. The helices arenumbered and the bound antagonist is shown as a space filling model.Amino acid residues in equivalent positions to the thermostabilisingmutations in the turkey β1 adrenergic receptor are shown as spacefilling models and are arrowed for clarity. Left: side view; right:angled top view.

FIG. 26 Comparison of the thermostabilities of three β adrenergicreceptors (turkey β1 (▪), human β1 (▾) and human β2 (●)) and twothermostabilised receptors (turkey β1-m23 (▴) and human β2-m23 (♦)). Thesix thermostabilising mutations in β1-m23 (R68S, M90V, Y227A, A282L,F327A, F338M) were all transferred directly to the human β2 receptor(K60S, M82V, Y219A, C265L, L310A, F321M) making β2-m23, based upon thealignment in FIGS. 9A-B. The resulting mutants were transientlyexpressed in mammalian cells, solubilised in 0.1% dodecylmaltoside andassayed for thermostability in the minus-ligand format (heating theapo-state, quenching on ice, adding 3H-DHA). The apparent Tms for turkeyβ1 and β2-m23 were 23° C. and 45° C. respectively, giving a ΔTm of 22°C. as seen previously in E. coli expressed receptor. The Tms for humanβ2 and β2-m23 were 29° C. and 41° C. respectively, showing that the aporeceptor was stabilised by 12° C. This exemplifies the principle of thetransferability of thermostabilising mutations from one receptor toanother receptor, which in this case are 59% identical. The human β1receptor (Tm˜12° C.) is much less stable than the turkey β1 receptor.

FIG. 27 Percentage identity of the turkey β1 adrenergic receptor, humanadenosine receptor and rat neurotensin receptor to human β adrenergicreceptors, human adenosine receptors and human neurotensin receptors,respectively.

FIGS. 28A-B Alignment of neurotensin receptors.

FIG. 29 shows a list of A2aR stabilising mutations. Mutants wereexpressed in E. coli, solubilised in 2% DDM+10% glycerol and tested forligand-binding, using the agonist [³H]-NECA and the antagonist[³H]-ZM241385. Concentrations of radioligands were 6-10-fold above theirK_(D) measured for the wild-type receptor. Expression of active receptorwas evaluated by ligand binding at 4° C. Stability was assayed byheating the solubilised receptor in its apo-state at 30° C. for 30minutes and then measuring residual binding activity. Under theseconditions, wild-type activity decays to 50% (S.D.=15%). Data obtainedfor expression and stability were normalised to wild-type values.Mutations included in subsequent rounds of mutagenesis were those whoseexpression was ≥30-40% and stability ≥130-140% compared to thewild-type. Bold lines indicate cluster of mutations.

EXAMPLE 1 Conformational Stabilisation of the β-Adrenergic Receptor inDetergent-Resistant Form

Summary

There are over 500 non-odorant G protein-coupled receptors (GPCRs)encoded by the human genome, many of which are predicted to be potentialtherapeutic targets, but there is only one structure available, that ofbovine rhodopsin, to represent the whole of the family. There are manyreasons for the lack of progress in GPCR structure determination, but wehypothesise that improving the detergent-stability of these receptorsand simultaneously locking them into one preferred conformation willgreatly improve the chances of crystallisation. A generic strategy forthe isolation of detergent-solubilised thermostable mutants of a GPCR,the β-adrenergic receptor, was developed based upon alanine scanningmutagenesis followed by an assay for receptor stability. Out of 318mutants tested, 15 showed a measurable increase in stability. Afteroptimisation of the amino acid residue at the site of each initialmutation, an optimally stable receptor was constructed by combiningspecific mutations. The most stable mutant receptor, βAR-m23, contained6 point mutations that led to a Tm 21° C. higher than the native proteinand, in the presence of bound antagonist, βARm23 was as stable as bovinerhodopsin. In addition, βAR-m23 was significantly more stable in a widerange of detergents ideal for crystallisation and was preferentially inan antagonist conformation in the absence of ligand.

Results

Selection of Single Mutations that Increase the Thermostability of theβ1 Adrenergic Receptor

βAR from turkey erythrocytes is an ideal target for structural studiesbecause it is well characterised and is expressed at high-levels ininsect cells using the baculovirus expression system[10,11]. The bestoverexpression of βAR is obtained using a truncated version of thereceptor containing residues 34-424 (βAR₃₄₋₄₂₄) [9] and this was used asthe starting point for this work. Alanine scanning mutagenesis was usedto define amino residues in βAR₃₄₋₄₂₄ that, when mutated, altered thethermostability of the receptor; if an alanine was present in thesequence it was mutated to a leucine residue. A total of 318 mutationswere made to amino acid residues 37-369, a region that encompasses allseven transmembrane domains and 23 amino acid residues at the Cterminus; mutations at 15 amino residues were not obtained due to strongsecondary structure in the DNA template. After sequencing each mutant toensure the presence of only the desired mutation, the receptors werefunctionally expressed in E. coli and assayed for stability.

The assay for thermostability was performed on unpurifieddetergent-solubilised receptors by heating the receptors at 32° C. for30 minutes, quenching the reaction on ice and then performing aradioligand binding assay, using the antagonist [³H]-dihydroalprenolol,to determine the number of remaining functional βAR₃₄₋₄₂₄ moleculescompared to the unheated control. Heating the unmutated βAR₃₄₋₄₂₄ at 32°C. for 30 min before the assay reduced binding to approximately 50% ofthe unheated control (FIG. 7 ); all the data for the mutants werenormalised by including the unmutated βAR₃₄₋₄₂₄ as a control in everyassay performed. In the first round of screening, eighteen mutantsshowed an apparent increase in stability, maintaining more than 75% ofantagonist binding after heating and being expressed in E. coli to atleast 50% of the native βAR₃₄₋₄₂₄ levels. In view of the possibility ofincreasing further the stability of these mutants, each of the 18residues was mutated to 2-5 alternative amino acid residues of varyingsize or charge (FIG. 1 ). Out of these 18 mutants, 12 were not improvedby further changes, 5 had better thermostability if another amino acidwas present and one mutation from the first screen turned out to be afalse positive. In addition, three residues that were not stabilisedupon mutation to alanine (V89, S151, L221) were mutated to a range ofother amino acid residues; the two positions that when mutated toalanine did not affect thermostability, were also unaffected by otherchanges. In contrast, V89 showed less thermostability when mutated toalanine, but thermostability increased when it was mutated to Leu. Thusthe initial alanine scanning successfully gave two-thirds of the bestamino acid residues of those tested for any given position.

The position and environment predicted for each of the 16 amino residuesthat gave the best increases in thermostability when mutated weredetermined by aligning the PAR sequence with that of rhodopsin whosestructure is known (FIG. 2 ). Fourteen of these residues were predictedto be present in transmembrane α-helices, with five of the residuespredicted to be lipid-facing, 4 being deeply buried and the remainderwere predicted to be at the interfaces between the helices. Some ofthese residues would be expected to interact with each other in the βARstructure, such as the consecutive amino acids G67 and R68 (V63 and Q64in rhodopsin), or the amino acids within the cluster Y227, R229, V230and A234 in helix 5 (Y223, Q225, L226 and V230 in rhodopsin). Otheramino acid residues that could interact in βAR were Q194A in externalloop 2 and D322A in external loop 3 (G182 and P285 in rhodopsin,respectively).

The increase in stability that each individual mutation gave toβAR₃₄₋₄₂₄ was determined by measuring the Tm for each mutant (resultsnot shown); Tm in this context is the temperature that gave a 50%decrease in functional binding after heating the receptor for 30minutes. Each mutation increased the Tm of βAR₃₄₋₄₂₄ by 1-3° C., withthe exception of M90A and Y227A that increased the Tm by 8° C.

Combining Mutations to Make an Optimally Stable Receptor

Initially, mutations that improved thermostability that were adjacent toone another in the primary amino sequence of βAR were combined.Constructions containing the mutations G67A and R68S, or differentcombinations of the mutations at the end of helix 5 (Y227A, R229Q, V230Aand A234L) were expressed and assayed; the Tm values (results not shown)were only 1-3° C. higher than the Tm for βAR₃₄₋₄₂₄ and one mutant wasactually slightly less stable, suggesting that combining mutations thatare adjacent to one another in the primary amino acid sequence does notgreatly improve thermostability. Subsequently, mutations predicted to bedistant from one another in the structure were combined. PCR reactionswere performed using various mixes of primers to combine up to 5different mutations in a random manner and then tested forthermostability (Table 1). The best of these combinations increased theTm more than 10° C. compared to the Tm of βAR₃₄₋₄₂₄. In some cases,there was a clear additive effect upon the Tm with the sequentialincorporation of individual mutations. This is seen in a series of 3mutants, m4-1, m4-7 and m4-2, with the addition of V230A to m4-1increasing the Tm by 2° C. and the additional mutation D332A in m4-7increasing the Tm a further 3° C. Mutants that contained Y227A and M90Aall showed an increase in Tm of 10° C. or more. Just these two mutationstogether increased the Tm of βAR₃₄₋₄₂₄ by 13° C. (m7-5), however, thetotal antagonist binding was less than 50% of βAR₃₄₋₄₂₄ suggestingimpaired expression of this mutant. The addition of F338M to m7-5 didnot increase the thermostability, but it increased levels of functionalexpression in E. coli.

TABLE 1 Combinations of mutations by PCR. PCR Receptor Mutations T_(m)(° C.) βAR₃₄₋₄₂₄ 31.7 ± 0.1 4 m4-1 G67A, G98A 35.5 ± 0.9 m4-2 G67A,G98A, V230A, D322A 40.9 ± 0.9 m4-6 G98A, D322A 35.0 ± 0.2 m4-7 G67A,G98A, V230A 38.0 ± 1.2 6 m6-1 Y227A, A234L, A282L, A334L 41.6 ± 0.9 m6-4R68S, Y227A, A234L, A282L 41.6 ± 0.1 m6-5 R68S, A234L, A282L, A334L 41.9± 0.5 m6-9 R68S, Y227A, A234L, A282L, A334L 43.7 ± 0.4 m6-10 R68S,Y227A, A282L, A334L 47.4 ± 1.1 m6-11 R68S, A282L, A334L 39.1 ± 0.5 7m7-1 M90V, A282L, F338M 43.0 ± 0.8 m7-2 M90V, A282L 38.9 ± 0.6 m7-5M90V, Y227A 45.2 ± 1.0 m7-6 M90V, I129V 40.0 ± 0.6 m7-7 M90V, Y227A,F338M 45.2 ± 2.0 10 m10-4 R68S, M90V, V230A, A334L 46.9 ± 1.0 m10-8R68S, M90V, V230A, F327A, A334L 47.3 ± 1.4 10 PCR reactions wereperformed combining different pairs of primers that contained theselected mutations. Successful PCR reactions are shown in the table. Thestability of these new mutants was assayed as described in FIG. 7 andthe Tm calculated. The results are shown as the mean ± S.E. fromduplicates.

The most thermostable mutants obtained, which were still expressed athigh levels in E. coli, were m6-10, m7-7 and m10-8. These mutantscontained collectively a total of 10 different mutations, with 8mutations occurring in at least two of the mutants. A second round ofmutagenesis was performed using m10-8 as the template and adding orreplacing mutations present in m6-10 and m7-7 (FIG. 3 ); some of thesemutations were very close in the primary amino acid sequence of βAR andtherefore were not additive as noted above, but many mutations improvedthe Tm further (Table 2). For example, exchanging two mutations inm10-8, to create m18, raised the Tm to 49.6° C. and adding A282L to makem23 increased the Tm a further 3° C. to 52.8° C. This produced the mostthermostable βAR₃₄₋₄₂₄ mutant so far and will be referred to as βAR-m23.

TABLE 2 Improvement of best combination of mutations. These new mutantswere obtained mixing the changes present in mutants m6-10, m7-7 andm10-8 by PCR. The stability of these new mutants was assayed asdescribed in FIG. 7 and the Tm calculated. The results are shown as themean ± S.E. from duplicates. Mutations T_(m) (° C.) m17 R68S M90V Y227AV230A — F327A A334L — 48.2 ± 1.4 m18 R68S M90V Y227A V230A A282L F327A —F338M 49.6 ± 0/9 m19 R68S M90V Y227A — A282L F327A — F338M 49.0 ± 0.8m20 R68S M90V — — — F327A A334L — 48.4 ± 0.7 m21 R68S M90V Y227A — —F327A A334L — 47.0 ± 1.3 m22 R68S M90V Y227A F327A A334L — 47.4 ± 0.5m23 R68S M90V Y227A — A282L F327A — F338M 52.8 ± 1.4

The thermostability assays used to develop βAR₃₄₋₄₂₄ mutants wereperformed by heating the receptor in the absence of the antagonist, butit is well known that bound ligand stabilises receptors. Therefore,stability assays for βAR₃₄₋₄₂₄ and βAR-m23 were repeated with antagonistbound to the receptors during the heating step (FIG. 4 ).

As expected, the Tm of the receptor that contained bound antagonistduring the incubation was higher than that for the receptor withoutantagonist. For βAR₃₄₋₄₂₄ the Tm was 6° C. higher with bound antagonistand for βAR-m23 the Tm increased 2° C. to 55° C.; the smaller increasein thermostability observed for βAR-m23 when antagonist binds suggeststhat the receptor is already in a more stable conformation similar tothe antagonist bound state than βAR₃₄₋₄₂₄ (see also below). The Tm ofβAR-m23 with antagonist bound is very similar to the Tm of dark-staterhodopsin in dodecylmaltoside (DDM)[12], whose structure has been solvedby two independent laboratories[13,14]. This suggested that βAR-m23 issufficiently stable for crystallisation.

Characterization of βAR-m23

The three characteristic activities measured for βAR-m23 and βAR₃₄₋₄₂₄to identify the effect of the six mutations were the affinity ofantagonist binding, the relative efficacies of agonist binding and theability of βAR-m23 to couple to G proteins. Saturation bindingexperiments to membranes using the antagonist [³H]-dihydroalprenolol(FIG. 8 ) showed that the affinity of binding to βAR-m23 (K_(D) 6.5±0.2nM, n=2) was slightly lower than for βAR₃₄₋₄₂₄ (K_(D) 2.8±0.1 nM, n=2),suggesting that there are no large perturbations in the structure ofβARm23 in the antagonist-bound conformation. This is consistent with theobservation that none of the mutations in βAR-m23 correspond with aminoacids believed to be implicated in ligand binding. In contrast toantagonist binding, the efficacy of agonist binding by βAR-m23 is 3orders of magnitude weaker than for βAR₃₄₋₄₂₄ (FIG. 5 ). The potency ofthe agonist isoprenaline is consistently lower in βAR-m23 and βAR₃₄₋₄₂₄than for the native agonist norepinephrine, indicating that theagonist-bound conformation for the two receptors is likely to besimilar. However, the large decrease in agonist efficacy in βAR-m23compared to βAR₃₄₋₄₂₄ indicates that the 6 mutations in βAR-m23 havelocked the receptor preferentially in an antagonist-bound conformation.From a crystallisation perspective, this is an added bonus tothermostabilisation, because it is essential to have a conformationallyhomogeneous protein population for the production of diffraction-qualitycrystals.

All of the thermostability assays used to derive βAR-m23 were performedon receptors solubilised in DDM. The aim of the thermostabilisationprocess was to produce a receptor that is ideal for crystallography,which means being stable in a variety of different detergents and notjust DDM. We therefore tested the stability of βAR-m23 and βAR in avariety of different detergents, concentrating on small detergents thatare preferentially used in crystallising integral membrane proteins.

Membranes prepared from E. coli expressing βAR-m23 or βAR₃₄₋₄₂₄ weresolubilised in DDM, bound to Ni-NTA agarose then washed with either DDM,decylmaltoside (DM), octylglucoside (OG), lauryldimethylamine oxide(LDAO) or nonylglucoside (NG). Stability assays were performed on thereceptors in each of the different detergents (FIG. 6 ). βAR₃₄₋₄₂₄ wasonly stable in DDM and DM, with no active receptors eluting from theresin washed with OG, NG or LDAO. In contrast, functional βAR-m23 wasstill present in all detergents and the Tm could be determined. Asexpected, the smaller detergents were considerably more denaturing thaneither DDM (Tm 52° C.) or DM (Tm 48° C.), with T_(m)s of 25° C. (NG),23° C. (LDAO) and 17° C. (OG). The difference in Tm between βAR-m23 andβAR₃₄₋₄₂₄ is about 20° C., irrespective of whether the receptors weresolubilised in either DDM or DM; it is therefore not surprising that noactive βAR₃₄₋₄₂₄ could be found in even NG, because the predicted Tmwould be about 5° C., thus resulting in rapid inactivation of thereceptor under the conditions used for purification. The selectionstrategy used for the generation of βAR-m23 was chosen deliberately tobe based upon thermostability, because it is far simpler to apply thanselecting for stability in detergents of increasing harshness. However,it is clear that increasing the thermostability of βAR₃₄₋₄₂₄ alsoresulted in increasing tolerance to small detergents ideal forcrystallising integral membrane proteins.

Crystallisation of Mutant GPCR

Earlier attempts to crystallise several different constructs of turkeybeta-adrenegic receptor failed. Despite experimenting with a variety ofconditions, using both the native sequence and several truncated andloop-deleted constructs, over many years, no crystals were obtained.

However, once the stabilising mutations from βAR-m23 were transferredinto the constructs, several different crystals were obtained indifferent detergents and different conditions.

The crystals that have been most studied so far were obtained using thepurified beta-36 construct (amino acid residues 34-367 of the turkeybeta receptor containing the following changes: point mutations C116Land C358A; the 6 thermostabilising point mutations in m23; replacementof amino acid residues 244-278 with the sequence ASKRK; a C terminalHis6 tag) expressed in insect cells using the baculovirus expressionsystem, after transferring the receptor into the detergentoctyl-thioglucoside. The precipitant used was PEG600 or PEG1000 and thecrystals obtained are elongated plates.

Experiments have also been carried out to see whether, once thecrystallisation conditions had been defined using the stabilisedreceptor, it was possible to get crystals using the originalnon-stabilised construct. It was possible that similar or perhaps verysmall crystals could have been obtained, but, in fact, the “wild type”(i.e. the starting structure from which the mutagenesis began) nevergave any crystals.

The crystals are plate-shaped with space group C2 and diffract well,though the cell dimensions do vary depending on the freezing conditionsused.

In general, once a GPCR has been stabilised it may be subjected to avariety of well-known techniques for structure determination. The mostcommon technique for crystallising membrane proteins is by vapourdiffusion (20, 21), usually using initially a few thousandcrystallisation conditions set up using commercial robotic devices (22).However, sometimes the crystals formed by vapour diffusion are small anddisordered, so additional techniques may then be employed. One techniqueinvolves the co-crystallisation (by vapour diffusion) of the membraneprotein with antibodies that bind specifically to conformationalepitopes on the proteins' surface (23, 24); this increases thehydrophilic surface of the protein and can form strong crystal contacts.A second alternative is to use a different crystallisation matrix thatis commonly called either lipidic cubic phase or lipidic mesophase (25,26), which has also been developed into a robotic platform (27). Thishas proven very successful for producing high quality crystals ofproteins with only small hydrophilic surfaces e.g. bacteriorhodopsin(28). Membrane protein structures can also be determined tohigh-resolution by electron crystallography (29).

The evolution of βAR-m23 from βAR₃₄₋₄₂₄ by a combination of alaninescanning mutagenesis and the selection of thermostable mutants hasresulted in a GPCR that is ideal for crystallography. The Tm for βAR-m23is 21° C. higher than for βAR₃₄₋₄₂₄ and, in the presence of antagonist,βAR-m23 has a similar stability to rhodopsin. The increased Tm ofβAR-m23 has resulted in an increased stability in a variety of smalldetergents that inactivate βAR₃₄₋₄₂₄. In addition, the selectionstrategy employed resulted in a receptor that is preferentially in theantagonist-bound conformation, which will also improve the chances ofobtaining crystals, because the population of receptor conformationswill be more homogeneous than for wild type βAR₃₄₋₄₂₄. Thus we haveachieved a process of conformational stabilisation in a single selectionprocedure.

It is not at all clear why the particular mutations we have introducedlead to the thermostabilisation of the receptor. Equivalent positions inrhodopsin suggest that the amino acid residues mutated could be pointinginto the lipid bilayer, into the centre of the receptor or at theinterfaces between these two environments. Given the difficulties intrying to understand the complexities of the thermostabilisation ofsoluble proteins[15], it seems unlikely that membrane proteins will beany easier to comprehend; we found that there was no particular patternin the amino acid residues in βAR that, when mutated, led tothermostability. However, since nearly 5% of the mutants produced weremore stable than the native receptor, alanine scanning mutagenesisrepresents an efficient strategy to rapidly identify thermostablemutants.

The procedure we have used to generate βAR-m23 is equally applicable toany membrane protein that has a convenient assay for detecting activityin the detergent solubilized form. While we have selected for stabilityas a function of temperature as the most convenient primary parameter,the procedure can easily be extended to test primarily for stability,for example, in a harsh detergent, an extreme of pH or in the presenceof chaotropic salts. Conformational stabilisation of a variety of humanreceptors, channels and transporters will make them far more amenable tocrystallography and will also allow the improvement in resolution ofmembrane proteins that have already been crystallised. It is to be hopedthat conformational stabilisation will allow membrane proteincrystallisation to become a far more tractable problem with a greaterprobability of rapid success than is currently the case. This shouldallow routine crystallisation of human membrane proteins in thepharmaceutical industry, resulting in valuable structural insights intodrug development.

Methods

Materials. The truncated β1 adrenergic receptor from turkey(βAR₃₄₋₄₂₄)[9] was kindly provided by Dr Tony Warne (MRC Laboratory ofMolecular Biology, Cambridge, UK). This βAR construct encoding residues34-424 contains the mutation C116L to improve expression[11], and aC-terminal tag of 10 histidines for purification.1-[4,6-propyl-³H]-dihydroalprenolol ([³H]-DHA) was supplied by AmershamBioscience, (+) L-norepinephrine bitartrate salt, (−) isoprenalinehydrochloride, (−) alprenolol tartrate salt and s-propranololhydrochloride were from Sigma.

Mutagenesis of DAR. The βAR cDNA was ligated into pRGIII to allow thefunctional expression of βAR in E. coli as a MalE fusion protein[16].Mutants were generated by PCR using the expression plasmid as templateusing the QuikChange II methodology (Stratagene). PCR reactions weretransformed into XL10-Gold ultracompetent cells (Stratagene) andindividual clones were fully sequenced to check that only the desiredmutation was present. Different mutations were combined randomly by PCRby including all the pairs of primers that introduced the followingmutations: Mut4, G67A, G068A, V230A, D322A and F327A; Mut6, R068S,Y227A, A234L, A282L and A334L; Mut7, M90V, I129V, Y227A, A282L andF338M; Mut10, R68S, M90V, V230A, F327A and A334L. The PCR mixes weretransformed and the clones sequenced to determine exactly whichmutations were introduced.

Protein expression and membrane preparations. Expression of βAR and themutants was performed in XL10 cells (Stratagene). Cultures of 50 ml of2×TY medium containing ampicillin (100 μg/ml) were grown at 37° C. withshaking until OD₆₀₀=3 and then induced with 0.4 mM IPTG. Inducedcultures were incubated at 25° C. for 4 h and then cells were harvestedby centrifugation at 13,000×g for 1 min (aliquots of 2 ml) and stored at−20° C. For the assays, cells were broken by freeze-thaw (five cycles),resuspended in 500 pa of buffer [20 mM Tris pH 8, 0.4 M NaCl, 1 mM EDTAand protease inhibitors (Complete™, Roche)]. After an incubation for 1 hat 4° C. with 100 μg/ml lysozyme and DNase I (Sigma), samples weresolubilized with 2% DDM on ice for 30 minutes. Insoluble material wasremoved by centrifugation (15,000×g, 2 min, 4° C.) and the supernatantwas used directly in radioligand binding assays.

For large-scale membrane preparations, 2L and 6L of E. coli culture ofβAR and Mut23, respectively, were grown as described above. Cells wereharvested by centrifugation at 5,000×g for 20 min, frozen in liquidnitrogen and stored at −80° C. Pellets were resuspended in 10 ml of 20mM Tris pH 7.5 containing 1× protease inhibitor cocktail (Complete™EDTA-free, Roche); 1 mg DNase I (Sigma) was added and the final volumewas made to 100 ml. Cells were broken by a French press (2 passages,20,000 psi), and centrifuged at 12,000×g for 45 min at 4° C. to removecell debris. The supernatant (membranes) was centrifuged at 200,000×gfor 30 min at 4° C.; the membrane pellet was resuspended in 15 ml of 20mM Tris pH 7.5 and stored in 1 ml aliquots at −80° C. afterflash-freezing in liquid nitrogen. The protein concentration wasdetermined by the amido black method[17]. These samples were used inradioligand binding assays after thawing and being solubilized in 2% DDMas above.

For competition assays, as well as testing different detergents,DDM-solubilized βAR was partially purified with Ni-NTA agarose (Qiagen).200 μl of Ni-NTA agarose was added to 2 ml of solubilized samples (10mg/ml of membrane protein) in 20 mM Tris pH 8, 0.4 M NaCl, 20 mMimidazole pH 8 and incubated for 1 h at 4° C. After incubation, sampleswere centrifuged at 13,000×g for 30 sec and washed twice with 250 μl ofbuffer (20 mM Tris pH 8, 0.4 M NaCl, 20 mM imidazole) containingdetergent (either 0.1% DDM, 0.1% DM, 0.1% LDAO, 0.3% NG or 0.7% OG).

Receptors were eluted in 2×100 μl of buffer (0.4 M NaCl, 1 mM EDTA, 250mM imidazole pH 8, plus the relevant detergent). The K_(D) for [³H]-DHAbinding to semipurified βAR₃₄₋₄₂₄ and βAR-m23 was, respectively 3.7 nMand 12.5 nM and the final concentration of [³H]-DHA used in thecompetition assays was 3 times the K_(D) ie 12 nM for βAR₃₄₋₄₂₄ and 40nM for βAR-m23.

Radioligand binding and thermostability assays. Single point bindingassays contained 20 mM Tris pH 8, 0.4 M NaCl, 1 mM EDTA, 0.1% DDM (orcorresponding detergent) with 50 nM [³H]-DHA and 20-100 μg membraneprotein in a final volume of 120 μl; equilibration was for 1 h at 4° C.Thermostability was assessed by incubating the binding assay mix, withor without [³H]-DHA at the specified temperature for 30 minutes;reactions were placed on ice and [³H]-DHA added as necessary andequilibrated for a further hour. Receptor-bound and free radioligandwere separated by gel filtration as described previously[18].Non-specific binding was determined in the presence of 1 μM ofs-propranolol. Saturation curves were obtained using a range of [³H]-DHAconcentration from 0.4 nM to 100 nM. Competition assays were performedusing a concentration of [³H]-DHA of 12 nM for βAR₃₄₋₄₂₄ and 40 nM forβAR-m23 (ie three times the K_(D)) and various concentrations ofunlabeled ligands (0-100 mM). Radioactivity was counted on a BeckmanLS6000 liquid scintillation counter and data were analyzed by nonlinearregression using Prism software (GraphPad).

Location of βAR-m23 thermostable mutations in rhodopsin structure. Thepdb file for the rhodopsin structure, accession code 1GZM[14], wasdownloaded from the Protein Data Bank website (www.pdb.org) anddisplayed in the program PyMOLX11Hybrid (DeLano Scientific). Theequivalent amino acid residues in rhodopsin for the thermostablemutations in βAR were located in the rhodopsin structure based upon analignment among the four GPCRs with which we are most familiar, namelyrhodopsin, β1 adrenergic receptor, neurotensin receptor and adenosineA_(2a) receptor[19].

EXAMPLE 2 Mutants of the Adenosine A_(2a) Receptor (A_(2a)R) withIncreased Thermostability

-   1. 315 site-directed mutants made between residues 2-316 of A_(2a)R.-   2. All of these mutants have been assayed for thermostability using    an assay measuring agonist and antagonist binding after the heating    step (Ligand(−) format as described in FIG. 12 ).    -   a. 26 mutants showed improved thermostability when measured with        ³H-NECA (agonist): G114 A, G118A, L167A, A184L, R199A, A203L,        L208A, Q210A, S213A, E219A, R220A, S223A, T224A, Q226A, K227A,        H230A, L241A, P260A, S263A, L267A, L272A, T279A, N284A, Q311A,        P313A, K315A.    -   b. 18 mutants showed improved thermostability when assayed with        ³H-ZM241385 (antagonist): A54L, V57A, H75A, T88A, G114A, G118A,        T119A, K122A, G123A, P149A, E151A, G152A, A203L, A204L, A231L,        L235A, V239A.-   3. Mutations have been combined to generate mutants in a putative    antagonist conformation. Wildtype A_(2a)R has a Tm of 31° C. with    ZM241385 bound.    -   a. Rant17 A54L+K122A+L235A Tm 48° C. (ZM241385 bound)    -   b. Rant19 A54L, T88A, V239A+A204L Tm 47° C. (ZM241385 bound)    -   c. Rant21 A54L, T88A, V239A+K122A Tm 49° C. (ZM241385 bound)-   4. Mutations from the agonist screen have been combined, but have    led to only a very low level of improvement in Tm of +2° C.

Table (i) in FIG. 29 provides a list of A2aR stabilising mutations.

TABLE (ii) Stability of best combinations. Tm (° C.) Tm (° C.) − + − +ago- ago- antago- antago- nist nist nist nist Wt 21 29 wt 31 32 Rag 1 2634 Rant 5 42 46 (A184L/R199A/ (A54L/T88A/ L272A) V239A) Rag 23 22 38Rant 21 41 49 (Rag 1 + F79A/ (Rant 5 + L208A) K122A) Receptors weresolubilised in 1% DDM (no glycerol). A melting profile was obtained byheating the solubilised receptor at different temperatures in absence(apo-state) or presence of ligand (ligand-occupied state). Data shownare representative of at least three independent experiments. S.D. is<1° C.

TABLE (iii) Summary of results for competition assays of detergent-solubilised wild-type A2aR and thermo-stable mutant Rant 21. K_(i) (M)Competitor wt Rant 21 XAC 2.3 × 10⁻⁶ 2.3 × 10⁻⁶ Theophylline 1.5 × 10⁻³0.9 × 10⁻³ NECA 7.0 × 10⁻⁶  >1 × 10⁻¹ R-PIA 1.6 × 10⁻⁵ 3.6 × 10⁻³ Valuesare representative of two independent experiments. Each data point wasassayed in triplicate and plotted as mean ± SD. Each solubilisedreceptor was incubated with ligands for one hour on ice in bindingbuffer (50 mM Tris pH 7.5 and 0.025% DDM) containing 400 mM NaCl.Binding of [3H]ZM241385 (10 nM) in the absence of unlabeled ligand wasset to 100%. Data shown are from two independent experiments with eachdata point measured in triplicate. Incubation of samples with ligandswas for 1 hour on ice with [³H]ZM241385 at a concentration of 10 nM.K_(i) values were calculated according to the Cheng and Prusoff equationusing the non-linear regression equation of the software Prism, applyinga K_(D) for [³H]ZM241385 of 12 nM for the wild-type and 15 nM for Rant21. Rant 21 did not bind NECA sufficiently for an accurate K_(i)determination (hence indicated as >1 × 10⁻¹). The affinity of Rant21 foragonist binding is weakened 232 fold for R-PIA and at least by 1900 foldfor NECA.

TABLE (iv) Summary of results for saturation assays ofdetergent-solubilised wild-type A2aR and thermo-stable mutants. K_(D)(nM) [³H]NECA [³H]ZM241385 Receptor (agonist) (antagonist) wt 32 ± 1 12± 3 Rag 1   26 ± 0.4   26 ± 0.5 Rag 23 21 ± 1 62 ± 1 Rant 21 >450 15 ± 3Values are representative of three independent experiments. Each datapoint was assayed in triplicate and plotted as mean ± SD. Data werefitted to the Michaelis-Menten equation using the non-linear regressionequation of the software Prism.

TABLE (v) Summary of stability of wild-type and mutant receptors indifferent detergents. Tm (° C.) Agonist-binding Antagonist-binding wtRag 23 wt Rant 21 0.01% DDM 27 34 25 39 0.1% DM 23 29 10 28 0.3% NM 2228 <4 25 0.3% NG † † † 22 0.6% OG <9 16 † 23 0.003% LDAO 28 38 32 420.006% FC12 37 39 43 49 Solubilisation of receptors and detergentexchange was performed during the IMAC step. S.D. is <1° C. It was notpossible to determine the Tm for some receptor-detergent combinations,because the receptor was too unstable (†).

EXAMPLE 3 Mutants of the Neurotensin Receptor (NTR) with IncreasedThermostability

-   1. 340 site-directed mutants have been made between residues 61-400    of NTR.-   2. Initially, all of these mutants were assayed for thermostability    using an assay measuring ³H-neurotensin (agonist) binding after the    heating step. 24 mutations led to a small but significant increase    in thermostability: A356L, H103A, D345A, A86L, A385L, Y349A, C386A,    K397A, H393A, 1116A, F358A, S108A, M181A, R392A, D113A, G209A,    L205A, L72A, A120L, P399A, Y351A, V268A, T207A, A155L, S362A, F189A,    N262A, L109A, W391A, T179A, S182A, M293A, L256A, F147A, D139A,    S100A, K176A, L111A, A90L, N270A.-   3. Mutants tested for thermostability by heating in the absence of    the agonist were re-tested using a slightly different assay where    the mutants were heated in the presence of ³H-neurotensin (Ligand(+)    format in FIG. 12 ). Mutants with improved thermostability are:    A69L, A73L, A86L, A90L, H103A, V165A, E166A, G215A, V229A, M250A,    I253A, A177L, R183A, I260A, T279A, T294A, G306A, L308A, V309A,    L310A, V313A, F342A, F358A, V360A, S362A, N370A, S373A, F380A,    A385L, P389A, G390A, R395A.-   4. There are also mutants that have a significantly enhanced    expression level compared to the wildtype receptor and could be used    to boost preceptor production levels for crystallisation: A86L,    H103A, F358A, S362A, N370A, A385L, G390A. All of these also have    increased thermostability.

5. Preferred combinations are

-   -   a. Nag7m F358A+A86L+I260A+F342A Tm 51° C. (neurotensin bound)    -   b. Nag7n F358A+H103A+I260A+F342A Tm 51° C. (neurotensin bound)        Wildtype NTR has a Tm of 35° C. with neurotensin bound.

EXAMPLE 4 Identification of Structural Motifs in which Stabilising GPCRMutations Reside

The structure of the β2 adrenergic receptor has been determined (20,21), which is 59% identical to the turkey β1 receptor, but with adistinctly different pharmacological profile (22, 23). In order todetermine the structural motifs in which the stabilising mutations ofthe turkey β1 receptor reside, we mapped the mutations onto the human β2structure (21).

The beta adrenergic receptors were first aligned using ClustalW in theMacVector package; thermostabilising mutations in turkey β1 werehighlighted along with the corresponding residue in the human β2sequence. The human β2 model (pdb accession code 2RH1) was visualised inPymol and the desired amino acids were shown as space filling models bystandard procedures known in the art. The structural motifs in which thestabilising mutations were located, were determined by visualinspection.

Table (vi) lists the equivalent positions in the β2 receptorcorresponding to the thermostabilising mutations in βAR-m23 and thestructural motifs in which they reside.

As seen from Table (vi), the mutations are positioned in a number ofdistinct localities. Three mutations are in loop regions that arepredicted to be accessible to aqueous solvent (loop). Eight mutationsare in the transmembrane α-helices and point into the lipid bilayer(lipid); three of these mutations are near the end of the helices andmay be considered to be at the hydrophilic boundary layer (lipidboundary). Eight mutations are found at the interfaces betweentransmembrane α-helices (helix-helix interface), three of which areeither within a kinked or distorted region of the helix (kink) andanother two mutations occur in one helix but are adjacent to one or moreother helices which contain a kink adjacent in space to the mutatedresidue (opposite kink). These latter mutations could affect the packingof the amino acids within the kinked region, which could result inthermostabilisation. Another mutation is in a substrate binding pocket.(pocket).

TABLE (vi) Position in the human β2 structure of the amino acid residuesequivalent to the thermostabilising mutations found in the turkey β1receptor and the structural motifs in which they reside. Turkey β1 Humanβ2 Description Helix 1 I55A I47 3-helix kink interface FIG. 18 Helix 1G67A A59 lipid boundary Helix 1 R68S K60 lipid boundary FIG. 25 Helix 2V89L V81 kink FIG. 19 Helix 2 M90V M82 kink FIG. 20 Helix 2 G98A G90pocket Helix 3 I129V I121 opposite kink FIG. 21 S151E S143 loop Helix 4V160A V152 lipid Q194A A186 loop Helix 5 L221V V213 lipid Helix 5 Y227AY219 helix-helix interface FIG. 23 Helix 5 R229Q R221 lipid Helix 5V230A V222 helix-helix interface Helix 5 A234L A226 helix-helixinterface Helix 6 A282L C265 loop FIG. 24 D322A K305 lipid boundaryHelix 7 F327A L310 lipid Helix 7 A334L V317 lipid Helix 7 F338M F321kink FIG. 22

Such structural motifs, by virtue of them containing stabilisingmutations, are important in determining protein stability. Therefore,targeting mutations to these motifs will facilitate the generation ofstabilised mutant GPCRs. Indeed, there were several instances where morethan one mutation mapped to the same structural motif. For example, theY227A, V230A and A234L mutations in the turkey β1 adrenergic receptorall mapped to the same helical interface, the V89L and M90V mutationsmapped to the same helical kink and the F327A and A334L mutations mappedto the same helical surface pointing towards the lipid bilayer (Table(vi)). Thus, when one stabilising mutation has been identified, thedetermination of the structural motif in which that mutation is locatedwill enable the identification of further stabilising mutations.

REFERENCES

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The invention claimed is:
 1. A mutant GPCR wherein (i) the mutant GPCRis a mutant β-adrenergic receptor which, when compared to thecorresponding wild-type adrenergic receptor, has increasedconformational thermostability and has at least one amino acidreplacement at a position which corresponds to any of the followingpositions according to the turkey β-adrenergic receptor defined as SEQID NO: 1: G67A, G98A, V230A, D322A, Y227A, A234L, A282L, A334L, R68S,M90V, F338M, I129V, V230A, or F327A; or wherein (ii) the mutant GPCR isa mutant adenosine receptor which, when compared to the correspondingwild-type adenosine receptor, has increased conformational stability andhas at least one amino acid replacement at a position which correspondsto any of the following positions according to the human adenosine A2areceptor defined as SEQ ID NO: 5: G114A, G118A, L167A, A184L, R199A,A203L, L208A, Q210A, S213A, E219A, R220A, S223A, T224A, Q226A, K227A,H230A, L241A, P260A, S263A, L267A, L272A, T279A, N284A, Q311A, P313A, orK315A; or wherein (iii) the mutant GPCR is a mutant neurotensin receptorwhich, when compared to the corresponding wild-type neurotensinreceptor, has increased conformational thermostability and has at leastone amino acid replacement at a position which corresponds to any of thefollowing positions according to the rat neurotensin receptor defined asSEQ ID NO: 9: A69L, A73L, A86L, A90L, H103A, V165A, E166A, G215A, V229A,M250A, I253A, A177L, R183A, I260A, T279A, T294A, G306A, L308A, V309A,L310A, V313A, F342A, F358A, V360A, S362A, N370A, S373A, F380A, A385L,P389A, G390A, or R395A.
 2. A mutant GPCR according to claim 1, which isin a solubilized form.
 3. A mutant GPCR according to claim 1, which isimmobilized to a solid support.